Compositions for inducing immune responses

ABSTRACT

The invention provides, inter alia, immunogenic compositions comprising a first antigen, at least two adjuvants, wherein a first adjuvant comprises a polymer derived from poly(lactides) and/or poly(lactide-co-glycolides), and wherein a second adjuvant comprises an imidazoquinoline, wherein said first antigen is encapsulated within, adsorbed or conjugated to, co-lyophilized or mixed with said first adjuvant, and a pharmaceutically acceptable excipient, wherein said composition elicits a cellular immune response when administered to a vertebrate subject. The invention also provides methods of producing immunogenic compositions, methods for producing a cytotoxic-T lymphocyte (CTL) response in a vertebrate subject, and methods of immunization.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority benefit of U.S. ProvisionalApplication 60/466,948, filed Apr. 30, 2003, which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to immunogenic agents and toagents which enhance the immune response to a selected antigen. Inparticular, the invention pertains to immunogenic compositionscomprising a first antigen, at least two adjuvants, wherein a firstadjuvant comprises a polymer derived from poly(lactides) and/orpoly(lactide-co-glycolides), and wherein a second adjuvant comprises animidazoquinoline, wherein said first antigen is encapsulated within,adsorbed or conjugated to, co-lyophilized or mixed with said firstadjuvant, and a pharmaceutically acceptable excipient. When administeredto a vertebrate subject the composition elicits a cellular immuneresponse.

BACKGROUND OF THE INVENTION

Numerous vaccine formulations have been developed which includeattenuated pathogens or subunit protein antigens. Conventional vaccinecompositions often include immunological adjuvants to enhancecell-mediated and humoral immune responses. For example, depot adjuvantsare frequently used which adsorb and/or precipitate administeredantigens and which can retain the antigen at the injection site. Typicaldepot adjuvants include aluminum compounds and water-in-oil emulsions.However, depot adjuvants, although increasing antigenicity, oftenprovoke severe persistent local reactions, such as granulomas, abscessesand scarring, when injected subcutaneously or intramuscularly. Otheradjuvants, such as lipopolysacharrides, can elicit pyrogenic responsesupon injection and/or Reiter's symptoms (influenza-like symptoms,generalized joint discomfort and sometimes anterior uveitis, arthritisand urethritis). Saponins, such as Quillaja saponaria, have also beenused as immunological adjuvants in vaccine compositions against avariety of diseases.

Complete Freund's adjuvant (CFA) is a powerful immunostimulatory agentthat has been successfully used with many antigens oh an experimentalbasis. CFA includes three components: a mineral oil, an emulsifyingagent, and killed mycobacteria, such as Mycobacterium tuberculosis.Although effective as an adjuvant, CFA causes severe side effectsprimarily due to the presence of the mycobacterial component, includingpain, abscess formation and fever. CFA, therefore, is not used in humanand veterinary vaccines.

Incomplete Freund's adjuvant (IFA) is similar to CFA but does notinclude the bacterial component. IFA, while not approved for use in theUnited States, has been used elsewhere in human vaccines for influenzaand polio and in veterinary vaccines for rabies, canine distemper andfoot-and-mouth disease. However, evidence indicates that both the oiland emulsifier used in IFA can cause tumors in mice.

Despite the presence of such adjuvants, conventional vaccines often failto provide adequate protection against the targeted pathogen. In thisregard, there is growing evidence that vaccination against intracellularpathogens, such as a number of viruses, should target both the cellularand humoral arms of the immune system. More particularly, cytotoxicT-lymphocytes (CTLs) play an important role in cell-mediated immunedefense against intracellular pathogens such as viruses andtumor-specific antigens produced by malignant cells. CTLs mediatecytotoxicity of virally infected cells by recognizing viral determinantsin conjunction with class I major histocompatibility complex (MHC)molecules displayed by the infected cells. Cytoplasmic expression ofproteins is a prerequisite for class I MHC processing and presentationof antigenic peptides to CTLs. However, immunization with killed orattenuated viruses often fails to produce the CTLs necessary to curbintracellular infection. Furthermore, conventional vaccinationtechniques against viruses displaying marked genetic heterogeneityand/or rapid mutation rates that facilitate selection of immune escapevariants, such as HIV or influenza, are problematic. Accordingly,alternative techniques for vaccination have been developed.

Particulate carriers with adsorbed or entrapped antigens have been usedin an attempt to circumvent these problems and in attempts to elicitadequate immune responses. Such carriers present multiple copies of aselected antigen to the immune system and promote trapping and retentionof antigens in local lymph nodes. The particles can be phagocytosed bymacrophages and can enhance antigen presentation through cytokinerelease. Examples of particulate carriers include those derived frompolymethyl methacrylate polymers, as well as polymer particles derivedfrom poly(lactides) and poly(lactide-co-glycolides), known as PLG. Whileoffering significant advantages over other more toxic systems,antigen-containing PLG particles to-date suffer from some drawbacks. Forexample, large-scale production and manufacturing of particulatecarriers may be problematic due to the high cost of the polymers used inthe manufacture the particulate carriers.

Liposomes have also been employed in an effort to overcome theseproblems. Liposomes are microscopic vesicles formed from lipidconstituents such as phospholipids which are used to entrappharmaceutical agents. Although the use of liposomes as a drug deliverysystem alleviates some of the problems described above, liposomesexhibit poor stability during storage and use, and large-scaleproduction and manufacturing of liposomes is problematic.

International Publication No. WO 98/50071 describes the use ofviral-like particles (VLPs) as adjuvants to enhance immune responses ofantigens administered with the VLPs. St. Clair et al. describe the useof protein crystals to enhance humoral and cellular responses. (St.Clair, N. et al., Applied Biol. Sci., 96:9469-9474, 1999).

Despite the above-described adjuvant and antigen-presentation systems,there is a continued need for effective, safe and cost-efficientcompositions with improved purity, stability and immunogenicity.

SUMMARY OF THE INVENTION

The inventors herein have found, surprisingly, that immunogeniccompositions comprising a first antigen, at least two adjuvants, whereina first adjuvant comprises a polymer derived from poly(lactides) and/orpoly(lactide-co-glycolides), and wherein a second adjuvant comprises animidazoquinoline, wherein said first antigen is encapsulated within,adsorbed or conjugated to, co-lyophilized or mixed with said firstadjuvant, and a pharmaceutically acceptable excipient, have aninhibitory effect on induction of antigen (Ag)-specific IFNγ-secretingcells, while having no inhibitory or stimulatory effect on the inductionof antigen-specific IL4-secreting cells. The present invention isdirected in some embodiments to the use of such compositions.

The present invention provides immunogenic compositions comprising afirst antigen, at least two adjuvants, wherein a first adjuvantcomprises a polymer derived from poly(lactides) and/orpoly(lactide-co-glycolides), and wherein a second adjuvant comprises animidazoquinoline, wherein said first antigen is encapsulated within,adsorbed or conjugated to, co-lyophilized or mixed with said firstadjuvant, and a pharmaceutically acceptable excipient, wherein saidcomposition elicits a cellular immune response when administered to avertebrate subject. In some embodiments the composition comprises animidazoquinoline.

In some embodiments, the present invention provides immunogeniccompositions comprising a first antigen, at least two adjuvants, whereina first adjuvant comprises a polymer derived from poly(lactides) and/orpoly(lactide-co-glycolides), wherein a second adjuvant comprises animidazoquinoline, and a pharmaceutically acceptable excipient, whereinthe first antigen is a particle produced by a process comprising thesteps of: (a) adding a precipitation agent to an aqueous solution of anantigen and stirring the resulting mixture to form the particle; (b)stabilizing the antigen particle by a stabilizing treatment; and (c)recovering the antigen particle from the aqueous solution.

In some embodiments, the present invention provides methods foreliciting a cytotoxic-T lymphocyte (CTL) response in a vertebratesubject comprising administering to the vertebrate subject animmunogenic composition of the present invention.

In some embodiments, the present invention provides methods of elicitingan immune response in a vertebrate subject comprising administering tothe vertebrate subject an effective amount of the immunogeniccomposition.

The present invention also provides injectable vaccine compositionscomprising immunogenic compositions of the present invention.

In some embodiments, the present invention provides methods of elicitingan antibody-mediated immune response in an individual comprisingadministering an immunogenic composition of the present invention to theindividual.

In some embodiments, the present invention provides methods of makingthe immunogenic compositions comprising: (a) combining a precipitationagent with an aqueous solution comprising an antigen; (b) dispersing theresultant mixture to form antigen particles; (c) stabilizing thedispersed antigen particles by a stabilizing treatment; (d) recoveringthe stabilized antigen particle; and (e) combining the stabilizedantigen particle with a pharmaceutically acceptable excipient.

In some embodiments, the present invention provides stabilized particlescapable of eliciting a cytotoxic-T lymphocyte (CTL) response, whereinthe stabilized particle is a generally spherical particle that isproduced by a process comprising: (a) forming a particle from antigen;(b) stabilizing the particle by a stabilizing treatment, wherein thestabilized particle is not a virus-like particle, and wherein thestabilized particle is not entrapped within a carrier; and (c) adding anadjuvant comprising R-848.

In some embodiments, the present invention provides methods of preparingan immunogenic composition comprising combining a stabilized particlewith one or more pharmaceutically acceptable excipients.

In some embodiments, the present invention provides pharmaceuticalcompositions comprising an immunogenic composition of the presentinvention.

In some embodiments, the present invention provides pharmaceuticalcompositions comprising a stabilized particle.

In some embodiments, the present invention provides kits for preparingan immunogenic composition comprising a first container comprising anantigen, a second container comprising an imidazoquinoline, and a thirdcontainer comprising a polymer derived from poly(lactides) and/orpoly(lactide-co-glycolides).

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows IgG and IgG2a antibody titers from mice immunized withgp120 associated with PLG plus R848 (2.5 μg); gp120 associated with PLGplus R848 (25 μg); gp120 associated with PLG plus R848 (2.5 μg)associated with PLG; gp120 associated with PLG plus R848 (25 μg)associated with PLG; gp120 associated with PLG and with R848 (2.5 μg);gp120 associated with PLG and with R848 (25 μg); gp120 and MF59 and R848(25 μg); gp120 associated with PLG, plus CpG; gp120 plus MF59; and gp120associated with PLG. Bars show the geometric mean antibody titer (GMT)of the group of mice. The error bars represent standard error of themean.

FIG. 2 shows levels of splenic cytokine secreting cells from miceimmunized with gp120 associated with PLG plus R848 (2.5 μg); gp120associated with PLG plus R848 (25 μg); gp120 associated with PLG plusR848 (2.5 μg) associated with PLG; gp120 associated with PLG plus R848(25 μg) associated with PLG; gp120 associated with PLG and with R848(2.5 μg); gp120 associated with PLG and with R848 (25 μg); gp120 andMF59 and R848 (25 μg); gp120 associated with PLG, plus CpG; gp120 plusMF59; and gp120 associated with PLG. Bars show the geometric mean cellnumber (GMT) of the group of mice. The error bars represent standarderror of the mean.

FIG. 3 shows antibody titers from mice immunized with: MenB Orf287associated with PLG; MenB Orf287 associated with PLG plus soluble R848(2.5 μg); MenB Orf287 associated with PLG plus soluble R848 (25 μg);MenB Orf287 associated with PLG plus soluble CpG (10 μg). Bars show thegeometric mean antibody titer (GMT) of the group of mice. The error barsrepresent standard error of the mean.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of virology, chemistry, biochemistry,recombinant technology, immunology and pharmacology, within the skill ofthe art. Such techniques are explained fully in the literature. See,e.g., Virology, 3rd Edition, vol. I & II (B. N. Fields and D. M. Knipe,eds., 1996); Remington's Pharmaceutical Sciences, 18th Edition (Easton,Pa.: Mack Publishing Company, 1990); Methods In Enzymology (S. Colowickand N. Kaplan, eds., Academic Press, Inc.); Handbook of ExperimentalImmunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986,Blackwell Scientific Publications); Sambrook et al., Molecular Cloning:A Laboratory Manual (2nd Edition, 1989); and DNA Cloning: A PracticalApproach, vol. I & II (D. Glover, ed.).

The present invention provides immunogenic compositions comprising afirst antigen, at least two adjuvants, wherein a first adjuvantcomprises a polymer derived from poly(lactides) and/orpoly(lactide-co-glycolides), and wherein a second adjuvant comprises animidazoquinoline, wherein said first antigen is encapsulated within,adsorbed or conjugated to, co-lyophilized or mixed with said firstadjuvant, and a pharmaceutically acceptable excipient, wherein saidcomposition elicits a cellular immune response when administered to avertebrate subject. In some embodiments the antigen is in the form of aparticle produced as described herein or by other means known to thoseof skill in the art. In some embodiments the antigen is a nucleic acidmolecule that is linked to a regulatory sequence which controlsexpression of the nucleic acid molecule. In some embodiments thecomposition comprises R-848 formulated with PLG.

The present invention also provides immunogenic composition comprising afirst antigen, at least two adjuvants, wherein a first adjuvantcomprises a polymer derived from poly(lactides) and/orpoly(lactide-co-glycolides), wherein a second adjuvant comprises animidazoquinoline, and a pharmaceutically acceptable excipient, whereinthe first antigen is a particle produced by a process comprising thesteps of: (a) adding a precipitation agent to an aqueous solution of anantigen and stirring the resulting mixture to form the particle; (b)stabilizing the antigen particle by a stabilizing treatment; and (c)recovering the antigen particle from the aqueous solution. In someembodiments, step (a) or the process to produce a particle (i.e. anantigen particle) can further include an acid. Examples of acidsinclude, but not limited to, acetic acid, glycolic acid, hydroxybutyricacid, hydrochloric acid or lactic acid. In some embodiments, the processof producing a particle includes a solvent evaporation technique.Solvent evaporation techniques are known to those of skill in the artand described herein. Examples of precipitation agents include, but arenot limited to, one or more of oils, hydrocarbons or coacervationagents.

The immunogenic compositions can further comprise at least a thirdadjuvant or more than 3, 4, or 5 additional adjuvants. In someembodiments, the immunogenic composition comprises R-848, MF59, orcombinations thereof.

The stabilizing treatment performed to prepare or make the particlescan, for example, comprise one or more of heat treatment or treatmentwith a chemical cross-linking agent. Processes of heat treatment orchemical-cross linking are described herein and known to one of ordinaryskill in the art.

In some embodiments, the compositions further comprise a second antigen.In some embodiments, the second antigen is distinct from the firstantigen. The second antigen is a nucleic acid molecule, protein, orpeptide.

In some embodiments, the particles described herein can be generallyspherical. In some embodiments, the particles or stabilized particleshave a diameter from about 200 nanometers to about 10 microns or fromabout 500 nanometers to about 5 microns.

Immunogenic compositions can also be used in methods for eliciting acytotoxic-T lymphocyte (CTL) response, antibody-mediated immuneresponse, or an immune response in a vertebrate subject comprisingadministering to the vertebrate subject the immunogenic composition.

In some embodiments, the immunogenic compositions are used in aninjectable vaccine to provide protection against a antigen, pathogen orprotein.

In some embodiments the present invention provides immunogeniccompositions comprising a first antigen, one or more adjuvants whereinat least one of said adjuvants comprises a polymer derived frompoly(lactides) and/or poly(lactide-co-glycolides), and apharmaceutically acceptable excipient, wherein at least one adjuvant isencapsulated within, adsorbed or conjugated to, co-lyophilized or mixedwith said first antigen, and wherein said composition elicits a cellularimmune response when administered to a vertebrate subject.

The present invention also provides methods of making immunogeniccompositions, the methods comprising (a) combining a precipitation agentwith an aqueous solution comprising an antigen; (b) dispersing theresultant mixture to form antigen particles; (c) stabilizing thedispersed antigen particles by a stabilizing treatment; (d) recoveringthe stabilized antigen particle; and (e) combining the stabilizedantigen particle with a pharmaceutically acceptable excipient. In someembodiments, the production of antigen particles further comprises asolvent evaporation technique. In some embodiments the compositioncomprises R-848 formulated with PLG.

Stabilized particles capable of eliciting a cytotoxic-T lymphocyte (CTL)response, wherein the stabilized particle is a generally sphericalparticle, can be produced by a process comprising: (a) forming aparticle from antigen; (b) stabilizing the particle by a stabilizingtreatment, wherein the stabilized particle is not a virus-like particle,and wherein the stabilized particle is not entrapped within a carrier;and (c) adding an adjuvant comprising R-848.

Immunogenic compositions can also be prepared as pharmaceuticalcompositions.

In some embodiments, the immunogenic compositions comprise conventionaladjuvants.

The present invention also provides kits for preparing an immunogeniccomposition. The kits comprise a first container comprising an antigenand a second container comprising a polymer derived from poly(lactides)and/or poly(lactide-co-glycolides). In some embodiments, the kitsfurther comprise a third container comprising R-848.

As used herein, the singular forms “a,” “an” and “the” include pluralreferences unless the content clearly dictates otherwise.

As used herein, the term “about” refers to +/−10% of a value.

The term “R-848” refers to a compound with the chemical name1H-Imidazo(4,5-c)quinoline-1-ethanol(ethoxymethyl)-Alpha, Alpha-dimethyland a chemical formula of C₁₇H₂₂N₄O₂. R-848 can also be referred to as“resiquimod,” “S-28463,” and “VML-600.” The synthesis of R-848 isdescribed in U.S. Pat. No. 5,389,640, herein incorporated by reference.R-848 is in a class of compounds referred to as imidazoquinolines whichhave cytokine induction activity. The antiviral and immunomodulatingactivities of R-848 has been discussed in Imbertson et al. (AntiviralResearch 26:A301, March 1995).

As used herein, the term “in combination with” is meant to refer to theuse of the compositions of the present invention with other therapeuticregimens. In some embodiments, immunogenic compositions of the presentinvention are used in combination with traditional treatment regimensfor diseases or disorders being treated. Administration of thecompositions of the present invention may take place prior to,simultaneously with, or after traditional treatments.

As used herein the term “isolated” refers to a polynucleotide, apolypeptide, an antibody, or a host cell that is in an environmentdifferent from that in which the polynucleotide, the polypeptide, or theantibody naturally occurs. Methods of isolating cells are well known tothose skilled in the art. A polynucleotide, a polypeptide, or anantibody which is isolated is generally substantially purified.

A “purified” protein is a protein which is recombinantly orsynthetically produced, or isolated from its natural host, such that theamount of protein present in a composition is substantially higher thanthat present in a crude preparation. In general, a purified protein willbe at least about 50% homogeneous, more preferably at least about 80%,about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, andabout 100% homogeneous.

An “immunological response” to an antigen or composition is thedevelopment in a subject of a humoral and/or a cellular immune responseto the antigen present in the composition of interest. For purposes ofthe present invention, a “humoral immune response” refers to an immuneresponse mediated by antibody molecules, while a “cellular immuneresponse” is one mediated by T-lymphocytes and/or other white bloodcells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (“CTL”s). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the MHC and expressed on the surfaces of cells. CTLshelp induce and promote the intracellular destruction of intracellularmicrobes, or the lysis of cells infected with such microbes. Anotheraspect of cellular immunity involves an antigen-specific response byhelper T-cells which act to help stimulate the function, and focus theactivity of, nonspecific effector cells against cells displaying peptideantigens in association with MHC molecules on their surface. A “cellularimmune response” also refers to the production of cytokines, chemokinesand other such molecules produced by activated T-cells and/or otherwhite blood cells, including those derived from CD4+ and CD8+ T-cells.

For purposes of the present invention, an “effective amount” of anantigen will be that amount which elicits an immunological response whenadministered, or enhances an immunological response to a coadministeredantigen.

As used herein, the phrase “injectable composition”, or variantsthereof, refers to compositions which satisfy the USP requirements for“injectables”, i.e., sterile, pyrogen- and particulate free, andpossessing specific pH and isotonicity values.

By “vertebrate subject” is meant any member of the subphylum chordata,including, without limitation, humans and other primates, includingnon-human primates such as chimpanzees and other apes and monkeyspecies; farm animals such as cattle, sheep, pigs, goats and horses;domestic mammals such as dogs and cats; laboratory animals includingrodents such as mice, rats and guinea pigs; birds, including domestic,wild and game birds such as chickens, turkeys and other gallinaceousbirds, ducks, geese, and the like. The term does not denote a particularage. Thus, both adult and newborn individuals are intended to becovered. The system described above is intended for use in any of theabove vertebrate species, since the immune systems of all of thesevertebrates operate similarly.

By “pharmaceutically acceptable” or “pharmacologically acceptable” ismeant a material which is not biologically or otherwise undesirable,i.e., the material may be administered to an individual along with theantigen formulation without causing any undesirable biological effectsor interacting in a deleterious manner with any of the components of thecomposition in which it is contained.

By “physiological pH” or a “pH in the physiological range” is meant a pHin the range of approximately 7.2 to 8.0 inclusive, more typically inthe range of approximately 7.2 to 7.6 inclusive.

As used herein, “treatment” refers to any of (i) the prevention ofinfection or reinfection, as in a traditional vaccine, (ii) thereduction or elimination of symptoms, and (iii) the substantial orcomplete elimination of the pathogen in question. Treatment may beeffected prophylactically (prior to infection) or therapeutically(following infection).

An “antigen” refers to a molecule containing one or more epitopes(either linear, conformational or both) that elicit an immunologicalresponse, as defined below. The term is used interchangeably with theterm “immunogen.” Normally, a B-cell epitope will include at least about5 amino acids but can be as small as 3-4 amino acids. A T-cell epitope,such as a CTL epitope, will include at least about 7-9 amino acids, anda helper T-cell epitope at least about 12-20 amino acids. The term“antigen” denotes both subunit antigens, i.e., antigens which areseparate and discrete from a whole organism with which the antigen isassociated in nature, as well as killed, attenuated or inactivatedbacteria, viruses, fungi, parasites or other microbes. Antibodies suchas anti-idiotype antibodies, or fragments thereof, and synthetic peptidemimotopes, which can mimic an antigen or antigenic determinant, are alsocaptured under the definition of antigen as used herein. Similarly, anoligonucleotide or polynucleotide which expresses an antigen orantigenic determinant in vivo, such as in gene therapy and DNAimmunization applications, is also included in the definition of antigenherein.

As used herein, the term “protein particle” refers to a particle madefrom a protein.

As used herein, the term “nucleic acid particle” refers to a particlemade from a nucleic acid molecule.

For purposes of the present invention, antigens can be derived from anyof several known viruses, bacteria, parasites and fungi, as described infurther detail below. The term also intends any of the various tumorantigens. For example, antigens may be proteins from or derived from theherpes virus family, including proteins derived from herpes simplexvirus (HSV) types 1 and 2, such as HSV-1 and HSV-2 glycoproteins gB, gDand gH; proteins derived from cytomegalovirus (CMV) including CMV gB andgH; proteins derived from hepatitis family of viruses, includinghepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus(HCV), the delta hepatitis virus (HDV), hepatitis E virus (HEV) andhepatitis G virus (HGV); proteins, including gp120, gp160, gp41, p24gagand p55gag envelope proteins, derived from HIV such as, includingmembers of the various genetic subtypes of HIV isolates HIV_(IIIb),HIV_(SF2), HIV_(LAV), HIV_(LAI), HIV_(MN), HIV-1_(CM235), HIV-1_(US4),HIV-2; proteins derived from simian immunodeficiency virus (SIV);proteins derived from Neisseria meningitidis (A, B, C, Y), Hemophilusinfluenza type B (HIB), Helicobacter pylori; human serum albumin andovalbumin. As discussed above, antigens may also be nucleic acids whichexpress an antigen or antigenic determinant in vivo.

As used herein, the term “PLG” refers to poly(lactide-co-glycolides),(see International Publications WO 00/06123 and WO 98/33487, each ofwhich is incorporated by reference in its entirety).

As used herein, “coacervation” refers to the phase separation of aliquid polymer-rich phase from a macromolecular solution when thesolubility is reduced by some chemical or physical means. The drug to beencapsulated is dispersed in a solution of a macromolecule in which itis immiscible. A non-solvent, miscible with the continuous phase but apoor solvent for the polymer under certain conditions will induce thepolymer to form a coacervate layer around the disperse phase. Thiscoating layer may then be treated to give a rigid coat. Examples ofcoacervation agents include, without limitation, acetone, ethanol,isopropanol, and the like.

As used herein the term “protein” refers to peptides, polypeptides,metalloproteins, glycoproteins and lipoproteins. “Proteins” refer topolymers of amino acid residues and are not limited to a minimum lengthof the product. Thus, peptides, oligopeptides, dimers, multimers, andthe like, are included within the definition. Both full-length proteinsand fragments thereof are encompassed by the definition. The terms alsoinclude modifications, such as deletions, additions and substitutions(generally conservative in nature), to the native sequence, so long asthe protein is capable of acting as an antigen and eliciting a CTLresponse.

Preferred substitutions are those which are conservative in nature,i.e., those substitutions that take place within a family of amino acidsthat are related in their side chains. Specifically, amino acids aregenerally divided into four families: (1) acidic-aspartate andglutamate; (2) basic-lysine, arginine, histidine; (3) non-polar-alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan; and (4) uncharged polar-glycine, asparagine, glutamine,cystine, serine threonine, tyrosine. Phenylalanine, tryptophan, andtyrosine are sometimes classified as aromatic amino acids. For example,it is reasonably predictable that an isolated replacement of leucinewith isoleucine or valine, an aspartate with a glutamate, a threoninewith a serine, or a similar conservative replacement of an amino acidwith a structurally related amino acid, will not have a major effect onthe biological activity. Proteins having substantially the same aminoacid sequence as the reference molecule, but possessing minor amino acidsubstitutions that do not substantially affect the immunogenicity of theprotein, are therefore within the definition of the referencepolypeptide.

The term “fragment” as used herein refers to a physically contiguousportion of the primary structure of a biomolecule. In the case ofproteins, a fragment is defined by a contiguous portion of the aminoacid sequence of that protein and refers to at least 3-5 amino acids, atleast 8-10 amino acids, at least 11-15 amino acids, at least 17-24 aminoacids, at least 25-30 amino acids, and at least 30-45 amino acids. Inthe case of polynucleotides, a fragment is defined by a contiguousportion of the nucleic acid sequence of that polynucleotide and refersto at least 9-15 nucleotides, at least 18-30 nucleotides, at least 33-45nucleotides, at least 48-72 nucleotides, at least 75-90 nucleotides, andat least 90-130 nucleotides. In some embodiments, fragments ofbiomolecules are immunogenic fragments.

In some embodiments, the antigen is a protein particle. Proteinparticles may have the following physical characteristics. The proteinparticles are generally spherical in shape and possess a diameter ofabout 150 nm to about 10 μm, about 200 nm to about 4 μm, and preferablyabout 250 nm to about 3 μm in diameter. Generally, the protein particlesare obtained by denaturing and cross-linking the protein, followed bystabilization of the cross-linked protein.

Examples of antigens useful in the present invention are set forth, forexample, in U.S. patent application Ser. No. 10/265,083, filed Oct. 3,2002, which is hereby incorporated by reference in its entirety.

Antigens useful in the present invention include for example and withoutlimitation, nucleic acids expressing proteins or proteins derived fromthe herpesvirus family, including proteins derived from herpes simplexvirus (HSV) types 1 and 2, such as HSV-1 and HSV-2 glycoproteins gB, gDand gH; antigens derived from varicella zoster virus (VZV), Epstein-Barrvirus (EBV) and cytomegalovirus (CMV) including CMV gB and gH; andantigens derived from other human herpesviruses such as HHV6 and HHV7.(See, e.g. Chee et al., Cytomegaloviruses (J. K. McDougall, ed.,Springer-Verlag 1990) pp. 125-169, for a review of the protein codingcontent of cytomegalovirus; McGeoch et al., J. Gen. Virol. (1988)69:1531-1574, for a discussion of the various HSV-1 encoded proteins;U.S. Pat. No. 5,171,568 for a discussion of HSV-1 and HSV-2 gB and gDproteins and the genes encoding therefor; Baer et al., Nature (1984)310:207-211, for the identification of protein coding sequences in anEBV genome; and Davison and Scott, J. Gen. Virol. (1986) 67:1759-1816,for a review of VZV.)

Antigens from the hepatitis family of viruses, including hepatitis Avirus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the deltahepatitis virus (HDV), hepatitis E virus (HEV) and hepatitis G virus(HGV), can also be conveniently used in the techniques described herein.By way of example, the viral genomic sequence of HCV is known, as aremethods for obtaining the sequence. See, e.g., International PublicationNos. WO 89/04669; WO 90/11089; and WO 90/14436, incorporated byreference herein in their entireties. The HCV genome encodes severalviral proteins, discussed further below. These proteins, as well asantigenic fragments thereof, will find use in the present methods.Similarly, the sequence for the .delta.-antigen from HDV is known (see,e.g., U.S. Pat. No. 5,378,814) and this sequence can also beconveniently used in the present methods. Additionally, antigens derivedfrom HBV, such as the core antigen, the surface antigen, sAg, as well asthe presurface sequences, pre-S1 and pre-S2 (formerly called pre-S), aswell as combinations of the above, such as sAg/pre-S1, sAg/pre-S2,sAg/pre-S1/pre-S2, and pre-S1/pre-S2, will find use herein. See, e.g.,“HBV Vaccines—from the laboratory to license: a case study” in Mackett,M. and Williamson, J. D., Human Vaccines and Vaccination, pp. 159-176,for a discussion of HBV structure; and U.S. Pat. Nos. 4,722,840,5,098,704, 5,324,513, incorporated herein by reference in theirentireties; Beames et al., J. Virol. (1995) 69:6833-6838, Birnbaum etal., J. Virol. (1990) 64:3319-3330; and Zhou et al., J. Virol. (1991)65:5457-5464.

Antigens derived from other viruses will also find use in the claimedmethods, such as without limitation, proteins from members of thefamilies Picornaviridae (e.g., polioviruses, etc.); Caliciviridae;Togaviridae (e.g., rubella virus, dengue virus, etc.); Flaviviridae;Coronaviridae; Reoviridae; Birnaviridae; Rhabodoviridae (e.g., rabiesvirus, etc.); Filoviridae; Paramyxoviridae (e.g., mumps virus, measlesvirus, respiratory syncytial virus, etc.); Orthomyxoviridae (e.g.,influenza virus types A, B and C, etc.); Bunyaviridae; Arenaviridae;Retroviradae (e.g., HTLV-I; HTLV-II; HIV-1 (also known as HTLV-III, LAV,ARV, hTLR, etc.)), including but not limited to antigens from theisolates HIV_(IIIb), HIV_(SV2), HIV_(LAV), HIV_(LAI), HIV_(MN));HIV-1_(CM235), HIV-1_(US4); HIV-2; simian immunodeficiency virus (SIV)among others. Additionally, antigens may also be derived from humanpapillomavirus (HPV) and the tick-borne encephalitis viruses. See, e.g.Virology, 3rd Edition (W. K. Joklik ed. 1988); Fundamental Virology, 2ndEdition (B. N. Fields and D. M. Knipe, eds. 1991), for a description ofthese and other viruses.

More particularly, the gp120 envelope protein from any of the above HIVisolates, including members of the various genetic subtypes of HIV, areknown and reported (see, e.g., Myers et al., Los Alamos Database, LosAlamos National Laboratory, Los Alamos, N. Mex. (1992); Myers et al.,Human Retroviruses and Aids, 1990, Los Alamos, N. Mex.: Los AlamosNational Laboratory; and Modrow et al., J. Virol. (1987) 61:570-578, fora comparison of the envelope gene sequences of a variety of HIVisolates) and sequences derived from any of these isolates will find usein the present methods. Furthermore, the invention is equally applicableto other immunogenic proteins derived from any of the various HIVisolates, including any of the various envelope proteins such as gp160,gp140 and gp41, gag antigens such as p24gag and p55gag, as well asproteins derived from the po1 region.

Influenza virus is another example of a virus for which the presentinvention is useful. Specifically, the envelope glycoproteins HA and NAof influenza A are of particular interest for generating an immuneresponse. Numerous HA subtypes of influenza A have been identified(Kawaoka et al., Virology (1990) 179:759-767; Webster et al., “Antigenicvariation among type A influenza viruses,” p. 127-168. In: P. Palese andD. W. Kingsbury (ed.), Genetics of influenza viruses. Springer-Verlag,New York). Thus, proteins derived from any of these isolates can also beused in the invention described herein.

Antigens for use in the compositions and methods described herein mayalso be derived from numerous bacterial antigens, such as those fromorganisms that cause diphtheria, cholera, tuberculosis, tetanus,pertussis, meningitis, and other pathogenic states, including, withoutlimitation, Meningococcus A, B and C, Hemophilus influenza type B (HIB),and Helicobacter pylori. Examples of parasitic antigens include thosederived from organisms causing malaria and Lyme disease.

Furthermore, the compositions and methods described herein provide ameans for treating a variety of malignant cancers. For example, thesystem of the present invention can be used to mount both humoral andcell-mediated immune responses to particular proteins specific to thecancer in question, such as an activated oncogene, a fetal antigen, oran activation marker. Such tumor antigens include any of the variousMAGEs (melanoma associated antigen E), including MAGE 1, 2, 3, 4, etc.(Boon, T. Scientific American (March 1993):82-89); any of the varioustyrosinases; MART 1 (melanoma antigen recognized by T cells), mutantras; mutant p53; p97 melanoma antigen; CEA (carcinoembryonic antigen),among others.

It is readily apparent that the present invention can be used to raiseantibodies to a large number of antigens for diagnostic andimmunopurification purposes, as well as to prevent or treat a widevariety of diseases.

As explained above, the compositions and methods of the presentinvention may employ HCV antigens. The genome of the hepatitis C virustypically contains a single open reading frame of approximately 9,600nucleotides, which is transcribed into a polyprotein. The full-lengthsequence of the polyprotein is disclosed in European Publication No.388,232 and U.S. Pat. No. 6,150,087, incorporated herein by reference intheir entireties. As shown in Table 1, An HCV polyprotein, uponcleavage, produces at least ten distinct products, in the order ofNH₂-Core-E 1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. The corepolypeptide occurs at positions 1-191, numbered relative to HCV-1 (see,Choo et al. (1991) Proc. Natl. Acad. Sci. USA 88:2451-2455, for theHCV-1 genome). This polypeptide is further processed to produce an HCVpolypeptide with approximately amino acids 1-173. The envelopepolypeptides, E1 and E2, occur at about positions 192-383 and 384-746,respectively. The P7 domain is found at about positions 747-809. NS2 isan integral membrane protein with proteolytic activity and is found atabout positions 810-1026 of the polyprotein. NS2, either alone or incombination with NS3 (found at about positions 1027-1657), cleaves theNS2—NS3 sissle bond which in turn generates the NS3 N-terminus andreleases a large polyprotein that includes both serine protease and RNAhelicase activities. The NS3 protease, found at about positions1027-1207, serves to process the remaining polyprotein. The helicaseactivity is found at about positions 1193-1657. Completion ofpolyprotein maturation is initiated by autocatalytic cleavage at theNS3—NS4a junction, catalyzed by the NS3 serine protease. SubsequentNS3-mediated cleavages of the HCV polyprotein appear to involverecognition of polyprotein cleavage junctions by an NS3 molecule ofanother polypeptide. In these reactions, NS3 liberates an NS3 cofactor(NS4a, found about positions 1658-1711), two proteins (NS4b found atabout positions 1712-1972, and NS5a found at about positions 1973-2420),and an RNA-dependent RNA polymerase (NS5b found at about positions2421-3011).

Sequences for HCV polyprotein products, and immunogenic polypeptidesderived therefrom, are known (see, e.g., U.S. Pat. No. 5,350,671,incorporated herein by reference in its entirety). For example, a numberof general and specific immunogenic polypeptides, derived from the HCVpolyprotein, have been described. See, e.g., Houghton et al., EuropeanPubl. Nos. 318,216 and 388,232; Choo et al. Science (1989) 244:359-362;Kuo et al. Science (1989) 244:362-364; Houghton et al. Hepatology (1991)14:381-388; Chien et al. Proc. Natl. Acad. Sci. USA (1992)89:10011-10015; Chien et al. J. Gastroent. Hepatol. (1993) 8:S33-39;Chien et al., International Publ. No. WO 93/00365; Chien, D. Y.,International Publ. No. WO 94/01778. These publications provide anextensive background on HCV generally, as well as on the manufacture anduses of HCV polypeptide immunological reagents. For brevity, therefore,the disclosure of these publications is incorporated herein byreference.

Any desired antigenic HCV polypeptide can be utilized with the presentinvention, including, for example, the E1 and/or E2 envelopeglycoproteins of HCV, as well as E1 E2 complexes, associated eitherthrough non-covalent or covalent interactions Such complexes may be madeup of immunogenic fragments of E1 and E2 which comprise epitopes. Forexample, fragments of E1 polypeptides can comprise from about 5 tonearly the full-length of the molecule, such as 6, 10, 25, 50, 75, 100,125, 150, 175, 185 or more amino acids of an E1 polypeptide, or anyinteger between the stated numbers. Similarly, fragments of E2polypeptides can comprise 6, 10, 25, 50, 75, 100, 150, 200, 250, 300, or350 amino acids of an E2 polypeptide, or any integer between the statednumbers. The E1 and E2 polypeptides may be from the same or differentHCV strains. For example, epitopes derived from, e.g., the hypervariableregion of E2, such as a region spanning amino acids 384-410 or 390-410,can be included in the E2 polypeptide. A particularly effective E2epitope to incorporate into the E2 sequence or E1 E2 complexes is onewhich includes a consensus sequence derived from this region, such asthe consensus sequence for amino acids 390-410 of the HCV type I genome.Additional epitopes of E1 and E2 are known and described in, e.g., Chienet al., International Publication No. WO 93/00365, incorporated byreference herein in its entirety.

Moreover, the E1 and E2 polypeptides may lack all or a portion of themembrane spanning domain. The membrane anchor sequence functions toassociate the polypeptide to the endoplasmic reticulum. Normally, suchpolypeptides are capable of secretion into growth medium in which anorganism expressing the protein is cultured. However, as described inInternational Publication No. WO 98/50556, such polypeptides may also berecovered intracellularly. Secretion into growth medium is readilydetermined using a number of detection techniques, including, e.g.,polyacrylamide gel electrophoresis and the like, and immunologicaltechniques such as immunoprecipitation assays as described in, e.g.,International Publication No. WO 96/04301, published Feb. 15, 1996. WithE1, generally polypeptides terminating with about amino acid position370 and higher (based on the numbering of HCV1 E1) will be retained bythe ER and hence not secreted into growth media. With E2, polypeptidesterminating with about amino acid position 731 and higher (also based onthe numbering of the HCV1 E2 sequence) will be retained by the ER andnot secreted. (See, e.g., International Publication No. WO 96/04301,published Feb. 15, 1996). It should be noted that these amino acidpositions are not absolute and may vary to some degree. Thus, thepresent invention contemplates the use of E1 and E2 polypeptides whichretain the transmembrane binding domain, as well as polypeptides whichlack all or a portion of the transmembrane binding domain, including E1polypeptides terminating at about amino acids 369 and lower, and E2polypeptides, terminating at about amino acids 730 and lower, areintended to be captured by the present invention. Furthermore, theC-terminal truncation can extend beyond the transmembrane spanningdomain towards the N-terminus. Thus, for example, E1 truncationsoccurring at positions lower than, e.g., 360 and E2 truncationsoccurring at positions lower than, e.g., 715, are also encompassed bythe present invention. All that is necessary is that the truncated E1and E2 polypeptides remain functional for their intended purpose.However, particularly preferred truncated E1 constructs are those thatdo not extend beyond about amino acid 300. Most preferred are thoseterminating at position 360. Preferred truncated E2 constructs are thosewith C-terminal truncations that do not extend beyond about amino acidposition 715. Particularly preferred E2 truncations are those moleculestruncated after any of amino acids 715-730, such as 725. If truncatedmolecules are used, it is preferable to use E1 and E2 molecules that areboth truncated.

E2 exists as multiple species (Spaete et al., Virol. (1992) 188:819-830;Selby et al., J. Virol. (1996) 70:5177-5182; Grakoui et al., J. Virol.(1993) 67:1385-1395; Tomei et al., J. Virol. (1993) 67:4017-4026) andclipping and proteolysis may occur at the N- and C-termini of the E1 andE2 polypeptides. Thus, an E2 polypeptide for use herein may comprise atleast amino acids 405-661, e.g., 400, 401, 402 . . . to 661, such as384-661, 384-715, 384-746, 384-749 or 384-809, or 384 to any C-terminusbetween 661-809, of an HCV polyprotein, numbered relative to thefull-length HCV-1 polyprotein. Similarly, preferable E1 polypeptides foruse herein can comprise amino acids 192-326, 192-330, 192-333, 192-360,192-363, 192-383, or 192 to any C-terminus between 326-383, of an HCVpolyprotein.

The E1 and E2 polypeptides and complexes thereof may also be present asasialoglycoproteins. Such asialoglycoproteins are produced by methodsknown in the art, such as by using cells in which terminal glycosylationis blocked. When these proteins are expressed in such cells and isolatedby GNA lectin affinity chromatography, the E1 and E2 proteins aggregatespontaneously. Detailed methods for producing these E1E2 aggregates aredescribed in, e.g., U.S. Pat. No. 6,074,852, incorporated herein byreference in its entirety. For example, E1E2 complexes are readilyproduced recombinantly, either as fusion proteins or by e.g.,co-transfecting host cells with constructs encoding for the E1 and E2polypeptides of interest. Co-transfection can be accomplished either intrans or cis, i.e., by using separate vectors or by using a singlevector which bears both of the E1 and E2 genes. If done using a singlevector, both genes can be driven by a single set of control elements or,alternatively, the genes can be present on the vector in individualexpression cassettes, driven by individual control elements. Followingexpression, the E1 and E2 proteins will spontaneously associate.Alternatively, the complexes can be formed by mixing the individualproteins together which have been produced separately, either inpurified or semi-purified form, or even by mixing culture media in whichhost cells expressing the proteins, have been cultured, if the proteinsare secreted. Finally, the E1E2 complexes of the present invention maybe expressed as a fusion protein wherein the desired portion of E1 isfused to the desired portion of E2.

Moreover, the E1 E2 complexes may be present as a heterogeneous mixtureof molecules, due to clipping and proteolytic cleavage, as describedabove. Thus, a composition including E1E2 complexes may include multiplespecies of E1 E2, such as E1E2 terminating at amino acid 746 (E1E2₇₄₆),E1E2 terminating at amino acid 809 (E1E2₈₀₉), or any of the othervarious E1 and E2 molecules described above, such as E2 molecules withN-terminal truncations of from 1-20 amino acids, such as E2 speciesbeginning at amino acid 387, amino acid 402, amino acid 403, etc.

E1 E2 complexes are readily produced recombinantly, either as fusionproteins or by e.g., co-transfecting host cells with constructs encodingfor the E1 and E2 polypeptides of interest. Co-transfection can beaccomplished either in trans or cis, i.e., by using separate vectors orby using a single vector which bears both of the E1 and E2 genes. Ifdone using a single vector, both genes can be driven by a single set ofcontrol elements or, alternatively, the genes can be present on thevector in individual expression cassettes, driven by individual controlelements. Following expression, the E1 and E2 proteins willspontaneously associate. Alternatively, the complexes can be formed bymixing the individual proteins together which have been producedseparately, either in purified or semi-purified form, or even by mixingculture media in which host cells expressing the proteins, have beencultured, if the proteins are secreted. Finally, the E1E2 complexes ofthe present invention may be expressed as a fusion protein wherein thedesired portion of E1 is fused to the desired portion of E2.

Methods for producing E1E2 complexes from full-length, truncated E1 andE2 proteins which are secreted into media, as well as intracellularlyproduced truncated proteins, are known in the art. For example, suchcomplexes may be produced recombinantly, as described in U.S. Pat. No.6,121,020; Ralston et al., J. Virol. (1993) 67:6753-6761, Grakoui etal., J. Virol. (1993) 67:1385-1395; and Lanford et al., Virology (1993)197:225-235.

Other HCV polypeptides may also be used in the invention. For example,HCV polypeptides derived from the Core region, such as polypeptidesderived from the region found between amino acids 1-191; amino acids10-53; amino acids 10-45; amino acids 67-88; amino acids 86-100; 81-130;amino acids 121-135; amino acids 120-130; amino acids 121-170; and anyof the Core epitopes identified in, e.g., Houghton et al., U.S. Pat. No.5,350,671; Chien et al. Proc. Natl. Acad. Sci. USA (1992)89:10011-10015; Chien et al. J. Gastroent. Hepatol. (1993) 8:S33-39;Chien et al., International Publ. No. WO 93/00365; Chien, D. Y.,International Publ. No. WO 94/01778; and U.S. Pat. No. 6,150,087, thedisclosures of which are incorporated herein by reference in theirentireties, will find use with the subject compositions and methods.

Additionally, polypeptides derived from the nonstructural regions of thevirus will also find use herein. The NS3/4a region of the HCVpolyprotein has been described and the amino acid sequence and overallstructure of the protein are disclosed in Yao et al. Structure (November1999) 7:1353-1363. See, also, Dasmahapatra et al., U.S. Pat. No.5,843,752, incorporated herein by reference in its entirety. Asexplained above, either the native sequence or immunogenic analogs canbe used in the subject formulations. Dasmahapatra et al., U.S. Pat. No.5,843,752 and Zhang et al., U.S. Pat. No. 5,990,276, both describeanalogs of NS3/4a and methods of making the same.

Moreover, polypeptides for use in the subject compositions and methodsmay be derived from the NS3 region of the HCV polyprotein. A number ofsuch polypeptides are known, including, but not limited to polypeptidesderived from the c33c and c100 regions, as well as fusion proteinscomprising an NS3 epitope, such as c25. These and other NS3 polypeptidesare useful in the present compositions and are known in the art anddescribed in, e.g., Houghton et al, U.S. Pat. No. 5,350,671; Chien etal. Proc. Natl. Acad. Sci. USA (1992) 89:10011-10015; Chien et al. J.Gastroent. Hepatol. (1993) 8:S33-39; Chien et al., International Publ.No. WO 93/00365; Chien, D. Y., International Publ. No. WO 94/01778; andU.S. Pat. No. 6,150,087, the disclosures of which are incorporatedherein by reference in their entireties.

Further, multiple epitope fusion antigens (termed “MEFAs”), as describedin International Publ. No. WO 97/44469, may be used in the subjectcompositions. Such MEFAs include multiple epitopes derived from two ormore of the various viral regions. The epitopes are preferably from morethan one HCV strain, thus providing the added ability to protect againstmultiple strains of HCV in a single vaccine.

It should be noted that for convenience, the various HCV regions aregenerally defined with respect to the amino acid number relative to thepolyprotein encoded by the genome of HCV-1a, as described in Choo et al.(1991) Proc Natl Acad Sci USA 88:2451, with the initiator methioninebeing designated position 1. However, the polypeptides for use with thepresent invention are not limited to those derived from the HCV-1asequence. Any strain or isolate of HCV can serve as the basis forproviding antigenic sequences for use with the invention. In thisregard, the corresponding regions in another HCV isolate can be readilydetermined by aligning sequences from the two isolates in a manner thatbrings the sequences into maximum alignment.

Various strains and isolates of HCV are known in the art, which differfrom one another by changes in nucleotide and amino acid sequence. Forexample, isolate HCV J1.1 is described in Kubo et al (1989) Japan. Nucl.Acids Res. 17:10367-10372; Takeuchi et al.(1990) Gene 91:287-291;Takeuchi et al. (1990) J. Gen. Virol. 71:3027-3033; and Takeuchi et al.(1990) Nucl. Acids Res. 18:4626. The complete coding sequences of twoindependent isolates, HCV-J and BK, are described by Kato et al., (1990)Proc. Natl. Acad. Sci. USA 87:9524-9528 and Takamizawa et al., (1991) J.Virol. 65:1105-1113, respectively. HCV-1 isolates are described by Chooet al. (1990) Brit. Med. Bull. 46:423-441; Choo et al. (1991) Proc.Natl. Acad. Sci. USA 88:2451-2455 and Han et al. (1991) Proc. Natl.Acad. Sci. USA 88:1711-1715. HCV isolates HC-J1 and HC-J4 are describedin Okamoto et al. (1991) Japan J. Exp. Med. 60:167-177. HCV isolates HCT18.about., HCT 23, Th, HCT 27, EC1 and EC10 are described in Weiner etal. (1991) Virol. 180:842-848. HCV isolates Pt-1, HCV-K1 and HCV-K2 aredescribed in Enomoto et al. (1990) Biochem. Biophys. Res. Commun.170:1021-1025. HCV isolates A, C, D & E are described inTsukiyama-Kohara et al. (1991) Virus Genes 5:243-254. HCV polypeptidesfor use in the compositions and methods of the invention can be obtainedfrom any of the above cited strains of HCV or from newly discoveredisolates isolated from tissues or fluids of infected patients.

Other antigens useful in the present invention are those derived fromHIV. The HIV genome includes the regions known as Gag (p55gag), Pol,Vif, Vpr, Tat, Rev, Vpu, Env and/or Nef. HIV antigens from any of theseregions, from any of the various subtypes, such as HIV subtype B and HIVsubtype C, as well as any of the various isolates will find use with thepresent methods. It will be readily apparent to one of ordinary skill inthe art in view of the teachings of the present disclosure how todetermine corresponding regions in other HIV strains or variants (e.g.,isolates HIV_(IIIb), HIV_(SF2), HIV-1_(SF162), HIV-1_(SF170), HIV_(LAV),HIV_(LAI), HIV_(MN), HIV-1_(CM235), HIV-1_(US4), other HIV-1 strainsfrom diverse subtypes (e.g., subtypes, A through G, and 0), HIV-2strains and diverse subtypes, and simian immunodeficiency virus (SIV).(See, e.g., Virology, 3rd Edition (W. K. Joklik ed. 1988); FundamentalVirology, 2nd Edition (B. N. Fields and D. M. Knipe, eds. 1991);Virology, 3rd Edition (Fields, B N, D M Knipe, P M Howley, Editors,1996, Lippincott-Raven, Philadelphia, Pa.; for a description of theseand other related viruses), using for example, sequence comparisonprograms (e.g., BLAST and others described herein) or identification andalignment of structural features (e.g., a program such as the “ALB”program described herein that can identify the various regions).

The envelope protein of HIV is a glycoprotein of about 160 kd (gp160).During virus infection of the host cell, gp160 is cleaved by host cellproteases to form gp120 and the integral membrane protein, gp41. Thegp41 portion is anchored in the membrane bilayer of virion, while thegp120 segment protrudes into the surrounding environment. gp120 and gp41are more covalently associated and free gp120 can be released from thesurface of virions and infected cells. The gp120 polypeptide isinstrumental in mediating entry into the host cell. Recent studies haveindicated that binding of CD4 to gp120 induces a conformational changein Env that allows for binding to a co-receptor (e.g, a chemokinereceptor) and subsequent entry of the virus into the cell. (Wyatt, R.,et al. (1998) Nature 393:705-711; Kwong, P., et al.(1998) Nature393:648-659). CD4 is bound into a depression formed at the interface ofthe outer domain, the inner domain and the bridging sheet of gp120.

Recombinant methods of obtaining the various HIV antigens once theregion desired is identified are well known in the art and are describedfurther below. See, also, U.S. Pat. No. 5,614,612, incorporated hereinby reference in its entirety.

Moreover, modified sequences of any of these HIV regions, such asmodified gp120 and p55gag, can be used in the subject methods. Sequencescan be modified for optimum codon usage to simulate human codons and toreduce toxicity. Such modified sequences are known in the art and thesequences and methods of producing the same are described in detail incommonly owned International Publication Nos. WO 00/39304 and WO00/39302, as well as in International Publication No. WO 98/34640, allincorporated herein by reference in their entireties.

The subject methods are also useful for antigens derived from Neisseriaspp., such as N. meningitidis, the causative agent of bacterialmeningitis and sepsis. Meningococci are divided into serological groupsbased on the immunological characteristics of capsular and cell wallantigens. Currently recognized serogroups include A, B, C, W-135, X, Y,Z and 29E. For purposes of the present invention, a meningococcalantigen may be derived from any of the various known serogroups. Thepolysaccharides responsible for the serogroup specificity have beenpurified from several of these groups, including A, B, C, W-135 and Y.Effective capsular polysaccharide-based vaccines have been developedagainst meningococcal disease caused by serogroups A, C, Y and W135 andany of these vaccine antigens will find use in the present compositionsand methods. See, e.g., International Publication Nos. WO 96/29412, WO96/14086, WO 99/57280, WO 00/22430, WO 99/24578, WO 99/36544, as well asTettelin et al. (2000) Science 287:1809-1815 and Pizza et al. (2000)Science 287:1816-1820, all incorporated by reference herein in theirentireties, for a description of various meningococcal protein antigensthat will find use herein. Additionally, saccharide antigens, such asthose from N. meningitidis serogroup A, C W135 and/or Y, such asdescribed in Costantino et al. (1992) Vaccine 10:691-698 and Costantinoet al. (1999) Vaccine 17:1251-1263 will find use herein. Other usefulNeisseria antigens include those derived from N. gonorrhorea, forexample, those described in International Publication Nos. WO 99/57280,WO 99/24578 and WO 99/36544.

For example, N. meningitidis serogroup B (termed “MenB” herein) accountsfor a large percentage of bacterial meningitis in infants and childrenresiding in the U.S. and Europe. Accordingly, antigens derived from MenBare particularly useful with the present compositions and methods, suchas any of the antigens expressed by the various open reading frames(ORFs) of the MenB genome. See, e.g., International Publication No. WO99/57280. Examples of such antigens include MenB proteins 961 and 287.Other meningococcal antigens for use herein include derivatives of thecapsular MenB polysaccharide (termed “MenB PS derivatives” herein).Examples of MenB PS derivatives are described in EP Publication No.504,202 B and U.S. Pat. No. 4,727,136. Also useful are molecularmimetics of unique epitopes of MenB PS as described in U.S. Pat. No.6,030,619. Additionally, outer membrane vesicle preparations from MenB,such as those described in International Patent ApplicationPCT/IB01/00166, Bjune et al. (1991) Lancet 338:1093-1096, Fukasawa etal. (1999) Vaccine 17:2951-2958 and Rosenquist et al. (1998) Dev. Biol.Stand. 92:323-333. All of the above references are incorporated hereinby reference in their entireties.

The complete genomic sequence of MenB, strain MC58, has been described.Tettelin et al., Science (2000) 287:1809. Several proteins that elicitedserum bactericidal antibody responses have been identified by wholegenome sequencing. Many of these proteins have sequences that are highlyconserved among Neisseria meningitidis. Pizza et al., Science (2000)287:1816. Accordingly, such antigens will find use in the presentinvention.

In some embodiments, proteins from which the protein particles areformed or the nucleic acids that encode the proteins from which nucleicacid particles are formed include, without limitation, viral proteins,fungal proteins, bacterial proteins, avian proteins, mammalian proteinsand eukaryotic proteins, such as but not limited to albumin, gelatin,zein, casein, collagen and fibrinogen. In some embodiments, proteinsfrom which the protein particles are formed include or the nucleic acidsthat encode the proteins from which nucleic acid particles are formedinclude, without limitation, proteins from the herpes virus family,including proteins derived from herpes simplex virus (HSV) types 1 and2, such as HSV-1 and HSV-2 glycoproteins gB, gD and gH; proteins derivedfrom cytomegalovirus (CMV) including CMV gB and gH; proteins derivedfrom hepatitis family of viruses, including hepatitis A virus (HAV),hepatitis B virus (HBV), hepatitis C virus (HCV), the delta hepatitisvirus (HDV), hepatitis E virus (HEV) and hepatitis G virus (HGV);proteins, including gp120, gp160, gp41, p24gag and p55gag envelopeproteins, derived from HIV such as, including members of the variousgenetic subtypes of HIV isolates HIV_(IIIb), HIV_(SF2), HIV_(LAV),HIV_(LAI), HIV_(MN), HIV-1_(CM235), HIV-1_(US4), HIV-2; proteins derivedfrom simian immunodeficiency virus (SIV); proteins derived fromNeisseria meningitidis (A, B, C, Y), Hemophilus influenza type B (HIB),Helicobacter pylori; human serum albumin and ovalbumin.

Methods for producing particular protein particles are known in the artand discussed more fully below. Furthermore, for purposes of the presentinvention, “antigen” refers to polynucleotides and proteins whichinclude modifications, such as deletions, additions and substitutions(generally conservative in nature), to the native sequence, so long asthe protein maintains the ability to elicit an immunological response,as defined herein. These modifications may be deliberate, as throughsite-directed mutagenesis, or may be accidental, such as throughmutations of hosts which produce the antigens.

Several detection techniques may be used in order to confirm thatproteins have taken on the conformation of protein particles. Suchtechniques include electron microscopy, X-ray crystallography, and thelike. See, e.g., Baker et al., Biophys. J. (1991) 60:1445-1456; Hagenseeet al., J. Virol. (1994) 68:4503-4505. For example, cryoelectronmicroscopy can be performed on vitrified aqueous samples of the proteinparticle preparation in question, and images recorded under appropriateexposure conditions.

An antigen particle is “distinct from” a second antigen when the secondantigen is not entrapped within the particles and/or the second antigenand particles are not expressed together as a fusion compound. However,an antigen particle is considered “distinct from” a selected secondantigen even if there is a loose physical association between the secondantigen and particle so long as the second antigen is not covalentlybound to, entrapped within or adsorbed to the surface of the particle.In some embodiments, the first or second antigen is a protein or anucleic acid molecule.

As used herein, the term “lysate” an extract or lysate derived from acell which includes one or more antigens. By way of example, an “H.pylori lysate” refers to an extract or lysate derived from an H. pyloriType I or Type II whole bacterium which includes one or more H. pyloriantigens. Thus, the term denotes crude extracts that contain severalantigens, as well as relatively purified compositions derived from suchcrude lysates which include only one or few such antigens. Such lysatesare prepared using techniques well known in the art.

Representative antigens that may be present in such lysates, eitheralone or in combination, include one or more antigens derived from theH. pylori adhesins such as, but not limited to, a 20 kDaα-acetyl-neuraminillactose-binding fibrillar haemagglutinin (HpaA), a 63kDa protein that binds phosphatidyl-ethanolamine and gangliotetraosylceramide, and a conserved fimbrial pilus-like structure. See, e.g.,Telford et al., Trends in Biotech. (1994) 12:420-426 for a descriptionof these antigens. Other antigens that may be present in the lysateinclude epitopes derived from any of the various flagellins such as themajor flagellin, FlaA and the minor flagellin, FlaB. In this regard, theflagella of H. pylori are composed of FlaA and FlaB, each with amolecular weight of approximately 53 kDa. Another representative antigenincludes H. pylori urease which is associated with the outer membraneand the periplasmic space of the bacterium. The holoenzyme is a largecomplex made up of two subunits of 26.5 kDa (UreA) and 61 kDa (UreB),respectively. Epitopes derived from the holoenzyme, either of thesubunits, or a combination of the three, can be present and are capturedunder the definition of “urease” herein. Another representative antigenthat may be present in the lysate or used in further purified formincludes the H. pylori heat shock protein known as “hsp60.” The DNA andcorresponding amino acid sequences for hsp60 are known. See, e.g.,International Publication No. WO 93/18150, published Sep. 16, 1993. Thefull-length hsp60 antigen shown has about 546 amino acids and amolecular weight of about 58 kDa. The VacA and CagA antigens may also bepresent in such lysates. It is to be understood that the lysate can alsoinclude other antigens not specifically described herein.

By “VacA antigen” is meant an antigen as defined above which is derivedfrom the antigen known as the H. pylori Type I Cytotoxin. The VacAprotein induces vacuolization in epithelial cells in tissue culture andcauses extensive tissue damage and ulceration when administered orallyto mice. The DNA and corresponding amino acid sequences for VacA areknown and reported in, e.g., International Publication No. WO 93/18150,published Sep. 16, 1993. The gene for the VacA antigen encodes aprecursor of about 140 kDa that is processed to an active molecule ofabout 90-100 kDa. This molecule, in turn, is proteolytically cleaved togenerate two fragments that copurify with the intact 90 kDa molecule.See, Telford et al., Trends in Biotech. (1994) 12:420-426. Thus, thedefinition of “VacA antigen” as used herein includes the precursorprotein, as well as the processed active molecule, proteolytic fragmentsthereof or portions or muteins thereof, which retain specific reactivitywith antibodies present in a biological sample from an individual withH. pylori Type I infection.

By “CagA antigen” is meant an antigen, as defined above, which isderived from the H. pylori Type I cytotoxin associated immunodominantantigen. CagA is expressed on the bacterial surface. The DNA andcorresponding amino acid sequences for CagA are known. See, e.g.,International Publication No. WO 93/18150, published Sep. 16, 1993. Thefull-length CagA antigen described therein includes about 1147 aminoacids with a predicted molecular weight of about 128 kDa. The nativeprotein shows interstrain size variability due to the presence of avariable number of repeats of a 102 bp DNA segment that encodes repeatsof a proline-rich amino acid sequence. See, Covacci et al., Proc. Natl.Acad. Sci. USA (1993) 90:5791-5795. Accordingly, the reported molecularweight of CagA ranges from about 120-135 kDa. Therefore, the definitionof “CagA antigen” as used herein includes any of the various CagAvariants, fragments thereof and muteins thereof, which retain theability to react with antibodies in a biological sample from anindividual with H. pylori Type I infection. For example, the CagApolypeptide depicted in FIG. 3 of Covacci et al. is a truncated proteinof 268 amino acids and includes Glu-748 to Glu-1015, inclusive, of thefull-length molecule. Further, the definition of “CagA antigen” as usedherein includes Nap protein of H. pylori antigen. See, e.g. PCTIB99/00695 for a description of Nap protein of H. pylori and methods topurify the same.

An immunogenic composition or vaccine that elicits a cellular immuneresponse may serve to sensitize a vertebrate subject by the presentationof antigen in association with MHC molecules at the cell surface. Thecell-mediated immune response is directed at, or near, cells presentingantigen at their surface. In addition, antigen-specific T-lymphocytescan be generated to allow for the future protection of an immunizedhost.

The ability of a particular antigen to stimulate a cell-mediatedimmunological response may be determined by a number of assays, such asby lymphoproliferation (lymphocyte activation) assays, CTL cytotoxiccell assays, or by assaying for T-lymphocytes specific for the antigenin a sensitized subject. Such assays are well known in the art. See,e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al.,Eur. J. Immunol. (1994) 24:2369-2376.

Thus, an immunological response as used herein may be one whichstimulates the production of CTLs, and/or the production or activationof helper T-cells. The antigen of interest may also elicit anantibody-mediated immune response. Hence, an immunological response mayinclude one or more of the following effects: the production ofantibodies by, e.g., but not limited to B-cells; and/or the activationof suppressor T-cells and/or γΔ T-cells directed specifically to anantigen or antigens present in the composition or vaccine of interest.These responses may serve to neutralize infectivity, and/or mediateantibody-complement, or antibody dependent cell cytotoxicity (ADCC) toprovide protection to an immunized host. Such responses can bedetermined using standard immunoassays and neutralization assays, wellknown in the art.

An immunogenic or vaccine composition which contains an antigen of thepresent invention, or an immunogenic or vaccine composition comprisingan adjuvant and/or a second antigen which is coadministered with thesubject antigen, displays “enhanced immunogenicity” when it possesses agreater capacity to elicit an immune response than the immune responseelicited by an equivalent amount of the antigen administered using adifferent delivery system, e.g., wherein the antigen is administered asa soluble protein. Thus, an immunogenic or vaccine composition maydisplay “enhanced immunogenicity” because the antigen is more stronglyimmunogenic or because a lower dose or fewer doses of antigen arenecessary to achieve an immune response in the subject to which theantigen is administered. Such enhanced immunogenicity can be determinedby administering the antigen composition and antigen controls to animalsand comparing antibody titers and/or cellular-mediated immunity againstthe two using standard assays described herein.

Combinations of antigens derived from the one or more organisms can beconveniently used to elicit immunity to multiple pathogens in a singlevaccine. An example of antigens in a multiple pathogen vaccine is acombination of bacterial surface oligosaccharides derived from MenC andHib, conjugated to a nontoxic mutant carrier derived from a bacterialtoxin, such as a nontoxic mutant of diphtheria toxin known as CRM₁₉₇.This conjugate is useful for preventing bacterial meningitis and isdescribed in International Publication No. WO 96/14086, published May17, 1996.

Methods and suitable conditions for forming particles from a widevariety of proteins are known in the art. For example, in the suspensioncross-linking process, a solution of a protein is added to an immiscibleliquid or an oil phase. The protein is dissolved in an appropriatesolvent, such as an alcohol (methanol, ethanol, isopropanol, and thelike), a ketone (methyl ethyl ketone, acetone, and the like), a glycol(ethylene glycol, propylene glycol, and the like) or an amide solvent(e.g., acetamide), containing between about 5% to about 90% of water. Aprecipitation agent is added to the protein solution form a proteinparticle. Oils such as mineral oil, silicone oil, or vegetable oil;hydrocarbons, such as hexane, heptane, dodecane, and high boilingpetroleum ether; and coacervation agents such as acetone, ethanol,isopropanol, and the like, are useful as precipitation agents. Theprotein particles are dispersed by high-speed stirring, and stabilizedusing stabilization treatment, such as heat treatment or by treatmentwith a chemical cross-linking agent. In particular, stabilization isachieved by heating of the suspension to a temperature about 30° C. toabout 150° C., preferably of about 35° C. to about 120° C., morepreferably of about 40° C. to about 100° C. Alternatively the proteinparticles are stabilized by treatment with a chemical cross-linkingagent, such as gluteraldehyde, butadione, and the like. See, e.g. WO96/10992; Polymers in Controlled Drug Delivery, Eds. Illum, L. andDavis, S. S. (Wright, 1987) Chapter 3, pg 25; Torrado, J. J. et al.,International Journal of Pharmaceutics, (1989) 51:85-93; Chen, G. Q. etal., Journal of Microencapsulation, (1994) 11(4):395-407.

In some embodiments, an aqueous solution of a protein, comprising about0.1 to about 20% protein solution, about 0.5 to about 10%, andpreferably about 1 to about 5% protein solution, is treated with anacid, until the pH is about 1 to about 6, about 1.5 to about 5, andpreferably about 2 to about 4, wherein the acid includes, but is notlimited to, acetic acid, glycolic acid, hydroxybutyric acid,hydrochloric acid, lactic acid, and the like. The solution is stirred athigh speed, about 1,000 to about 25,000 rpm, about 2,000 to about15,000, and preferably about 5,000 to about 10,000 rpm for about 1minute to about 60 minutes, about 5 to about 45 minutes, and preferablyabout 10 to about 30 minutes. A coacervation agent is added to thestirring solution to form the protein particles, and the mixture isstirred for about 1 minute to about 60 minutes, about 5 to about 45minutes, and preferably about 10 to about 30 minutes. Coacervationagents include, but are not limited to acetone, ethanol, isopropanol,and the like. The coacervation agent is optionally evaporated and theprotein particles are stabilized by heating the mixture at about 30 toabout 70° C., about 35 to about 65° C., and preferably about 40 to about60° C., for about 1 minute to about 60 minutes, about 5 to about 45minutes, and preferably about 10 to about 30 minutes, with stirring atabout 1,000 to about 25,000 rpm, about 2,000 to about 15,000, andpreferably about 5,000 to about 10,000 rpm. The protein particles may besized, for example, in a Malvern Master sizer.

In some embodiments, an aqueous solution of the antigen, as describedabove, is added to a precipitation agent, such as mineral oil, siliconeoil, or vegetable oil, and/or hydrocarbons, such as hexane, heptane,dodecane, and high boiling petroleum ether. The emulsion is stirred athigh speed, about 1,000 to about 25,000 rpm, about 2,000 to about15,000, and preferably about 5,000 to about 10,000 rpm for about 1minute to about 60 minutes, about 5 to about 45 minutes, and preferablyabout 10 to about 30 minutes. The mixture is heated at about 30 to about70° C., about 35 to about 65° C., and preferably about 40 to about 60°C., for about 1 minute to about 60 minutes, about 5 to about 45 minutes,and preferably about 10 to about 30 minutes, with stirring at about1,000 to about 25,000 rpm, about 2,000 to about 15,000, and preferablyabout 5,000 to about 10,000 rpm to stabilize the antigen particles. Themixture is centrifuged and the antigen particles are collected. Theantigen particles may be sized, for example, in a Malvern Master sizer.

Once obtained, antigen particles can be incorporated into immunogenic orvaccine compositions optionally comprising an adjuvant and/or a selectedsecond antigen. The adjuvant and/or the second antigen can beadministered separately, either simultaneously with, prior to, orsubsequent to, the administration of the antigen particle composition.The compositions can be used both for treatment and/or prevention ofinfection. Furthermore, the formulations of the invention comprisingprotein particles may be used to enhance the activity of selected secondantigens produced in vivo, i.e., in conjunction with DNA immunization.

Antigen particles can be used in compositions for immunizing avertebrate subject against one or more pathogens or against subunitantigens derived from pathogens, or for priming an immune response toone or several antigens. Antigens that can be administered as a secondantigen with the antigen particle include proteins, polypeptides,antigenic protein fragments, oligosaccharides, polysaccharides, and thelike. Similarly, an oligonucleotide or polynucleotide, encoding adesired antigen, can be administered with the antigen particle for invivo expression.

As explained above, antigen particle formulations may or may not containa second antigen of interest. For example, antigen particles may beformed from a combination of an appropriate nucleic acid molecule orprotein and an antigen, or the antigens can be administered separatelyfrom the protein particle compositions at the same or at differentsites. In any event, one or more selected antigens will be administeredin a “therapeutically effective amount” such that an immune response canbe generated in the individual to which it is administered. The exactamount necessary will vary depending on the subject being treated; theage and general condition of the subject to be treated; the capacity ofthe subject's immune system to synthesize antibodies and/or mount acell-mediated immune response; the degree of protection desired; theseverity of the condition being treated; the particular antigen selectedand its mode of administration, among other factors. An appropriateeffective amount can be readily determined by one of skill in the art.Thus, a “therapeutically effective amount” will fall in a relativelybroad range that can be determined through routine trials. In general, a“therapeutically effective” amount of antigen will be an amount on theorder of about 0.1 μg to about 1000 μg, preferably about 1 μg to about100 μg.

Similarly, antigens will be present in an amount such that a secondantigen, if present, displays “enhanced immunogenicity,” as definedabove. Amounts which are effective for eliciting an enhanced immuneresponse can be readily determined by one of skill in the art.

In some embodiments each adjuvant is present in the amount of about0.06% to about 1% w/v, from about 0.1% to about 0.6% w/v, or 0.01% toabout 2% w/v.

The compositions may additionally contain one or more “pharmaceuticallyacceptable excipients or vehicles” such as water, saline, glycerol,ethanol, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, biological buffers, and the like, may be present insuch vehicles. A biological buffer can be virtually any solution whichis pharmacologically acceptable and which provides the adjuvantformulation with the desired pH, i.e., a pH in the physiological range.Examples of buffer solutions include saline, phosphate buffered saline,Tris buffered saline (TBS), Hank's buffered saline (HBS), growth mediasuch as Eagle's Minimum Essential Medium (“MEM”), and the like.

The second antigen is optionally associated with a carrier (e.g., theantigen may be encapsulated within, adsorbed on to, co-lyophilized ormixed with the carrier), wherein the carrier is a molecule that does notitself induce the production of antibodies harmful to the individualreceiving the composition. Suitable carriers are typically large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,lipid aggregates (such as oil droplets or liposomes), polymericparticulate carriers, inactive virus particles and the like.Additionally, these carriers may function as additionalimmunostimulating agents. Furthermore, the antigen may be conjugated toa bacterial toxoid, such as toxoid from diphtheria, tetanus, cholera,etc. Examples of polymeric particulate carriers include particulatecarriers formed from materials that are sterilizable, non-toxic andbiodegradable. Such materials include, without limitation,poly(α-hydroxy acid), polyhydroxybutyric acid, polycaprolactone,polyorthoester and polyanhydride. Preferably, microparticles for usewith the present invention are derived from a poly(a-hydroxy acid), inparticular, from a poly(lactide) (“PLA”) or a copolymer of D,L-lactideand glycolide or glycolic acid, such as a poly(D,L-lactide-co-glycolide)(“PLG” or “PLGA”), or a copolymer of D,L-lactide and caprolactone. Themicroparticles may be derived from any of various polymeric startingmaterials which have a variety of molecular weights and, in the case ofthe copolymers such as PLG, a variety of lactide:glycolide ratios, theselection of which will be largely a matter of choice, depending in parton the coadministered second antigen (for a further discussion ofparticulate carriers for use herein, see commonly owned, U.S. patentapplication Ser. No. 09/124,533, filed on Jul. 29, 1998).

The adjuvant/second antigen may be conjugated on to the surface of theantigen using any of the several methods known in the art (see, e.g.,Bioconjugate Techniques, Greg. T. Hermanson Ed., Academic Press, NewYork. 1996). For example, protein-protein (i.e. protein particle-secondantigen) conjugation could be carried by using sulfo-SMCC linkers(sulfosuccinimidyl esters) for conjugation using standard protocols.

Adjuvants may also be used to enhance the effectiveness of thepharmaceutical compositions. Such adjuvants include, but are not limitedto: (1) aluminum salts (alum), such as aluminum hydroxide, aluminumphosphate, aluminum sulfate, etc.; (2) oil-in-water emulsionformulations (with or without other specific immunostimulating agentssuch as muramyl peptides (see below) or bacterial cell wall components),such as, for example; (a) MF59 (International Publication No. WO90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85(optionally containing various amounts of MTP-PE (see below), althoughnot required) formulated into submicron particles using a microfluidizersuch as Model 110Y microfluidizer (Microfluidics, Newton, Mass.); (b)SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymerL121, and thr-MDP (see below) either microfluidized into a submicronemulsion or vortexed to generate a larger particle size emulsion, and;(c) Ribi™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.)containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cellwall components from the group consisting of monophosphorylipid A (MPL),trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferablyMPL+CWS (Detox™) (for a further discussion of suitable submicronoil-in-water emulsions for use herein, see International Publication No.WO 99/30739, published Jun. 24, 1999); (3) saponin adjuvants, such asStimulon™ (Cambridge Bioscience, Worcester, Mass.) may be used orparticles generated therefrom such as ISCOMs (immunostimulatingcomplexes); (4) Complete Freunds Adjuvant (CFA) and Incomplete FreundsAdjuvant (IFA); (5) cytokines, such as interleukins (IL-1, IL-2, IL-3,IL-4, IL-S, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-16,IL-17, IL-19, IL-20, and the like), macrophage colony stimulating factor(M-CSF), tumor necrosis factor (TNF), VEGF, CD27, CD30, CD40, FasLigand, Placenta Growth Factor, etc.; (6) detoxified mutants of abacterial ADP-ribosylating toxin such as a cholera toxin (CT), apertussis toxin (PT), or an E. coli heat-labile toxin (LT), particularlyLT-K63 (where lysine is substituted for the wild-type amino acid atposition 63) LT-R72 (where arginine is substituted for the wild-typeamino acid at position 72), CT-S 109 (where serine is substituted forthe wild-type amino acid at position 109), adjuvants derived from theCpG family of molecules, CpG dinucleotides and syntheticoligonucleotides which comprise CpG motifs (see, e.g., Krieg et al.,Nature, 374:546 (1995) and Davis et al., J. Immunol., 160:870-876(1998)) and PT-K9/G129 (where lysine is substituted for the wild-typeamino acid at position 9 and glycine substituted at position 129) (see,e.g., International Publication Nos. WO93/13202 and WO92/19265); (7)R-848 (see e.g., U.S. Pat. Nos. 5,352,784; 5,266,575; 4,929,624;5,268,376; 5,389,640; 4,689,338; 5,482,936; 5,346,905; 5,395,937;5,238,944; 5,252,612, and 6,110,929; and international publicationWO99/29693); and (8) other substances that act as immunostimulatingagents to enhance the effectiveness of the composition.

As used herein, the term “conventional adjuvants” refers to adjuvantsthat have been known and used previously in vaccine compositions,immunogenic compositions, or in compositions that have been used togenerate an immune response. Examples of conventional adjuvants include,but are not limited to, aluminum salts (alum), oil-in-water emulsionformulations (with or without other specific immunostimulating agentssuch as muramyl peptides (see below) or bacterial cell wall components),such as for example (a) MF59 (International Publication No. WO90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85(optionally containing various amounts of MTP-PE (see below), althoughnot required) formulated into submicron particles using a microfluidizersuch as Model 110Y microfluidizer (Microfluidics, Newton, Mass.), (b)SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymerL121, and thr-MDP (see below) either microfluidized into a submicronemulsion or vortexed to generate a larger particle size emulsion, and(c) Ribi™ adjuvant system (RAS) containing 2% Squalene, 0.2% Tween 80,and one or more bacterial cell wall components from the group consistingof monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS), preferably MPL+CWS (Detox™), saponin adjuvants, such asStimulon™ or particle generated therefrom such as ISCOMs(immunostimulating complexes), Complete Freunds Adjuvant (CFA) andIncomplete Freunds Adjuvant (IFA), cytokines, such as interleukins(IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, IL-16, IL-17, IL-19, IL-20, and the like), macrophagecolony stimulating factor (M-CSF), tumor necrosis factor (TNF), VEGF,CD27, CD30, CD40, Fas Ligand, Placenta Growth Factor, etc.; detoxifiedmutants of a bacterial ADP-ribosylating toxin such as a cholera toxin(CT), a pertussis toxin (PT), or an E. coli heat-labile toxin (LT),particularly LT-K63, LT-R72, CT-S109, adjuvants derived from the CpGfamily of molecules, CpG dinucleotides and synthetic oligonucleotideswhich comprise CpG motifs, and PT-K9/G129.

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipahitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

Once formulated, the compositions of the invention can be administeredparenterally, e.g., by injection. The compositions can be injectedeither subcutaneously, intraperitoneally, intravenously orintramuscularly. Other modes of administration include oral andpulmonary administration, suppositories, mucosal and transdermalapplications. Dosage treatment may be a single dose schedule or amultiple dose schedule. A multiple dose schedule is one in which aprimary course of vaccination may be with 1-10 separate doses, followedby other doses given at subsequent time intervals, chosen to maintainand/or reinforce the immune response, for example at 1-4 months for asecond dose, and if needed, a subsequent dose(s) after several months.The dosage regimen will also, at least in part, be determined by theneed of the subject and be dependent on the judgment of thepractitioner. Furthermore, if prevention of disease is desired, thevaccines are generally administered prior to primary infection with thepathogen of interest. If treatment is desired, e.g., the reduction ofsymptoms or recurrences, the vaccines are generally administeredsubsequent to primary infection.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Example 1

Preparation of Small Ovalbumin (OVA) Protein Particles

Ovalbumin (OVA, 200 mg) was dissolved in distilled water (10 ml) to forma 2% protein solution. Lactic acid (100 μl) was added to theOVA-solution until the pH was reduced to about 4.5-5.0. The solution wasstirred over a magnetic stirrer at 1500 rpm for 10 minutes. Acetone (25ml) was added to the stirring solution, and the mixture was leftstirring for 10 minutes. The mixture was heated at 70° C. for 30 minuteswith stirring at 5000 rpm to stabilize the protein particles. Theprotein particles were then sized in a Malvern Master sizer for futureuse (the protein particles were about 250 nm in diameter).

Example 2

Preparation of Large Ovalbumin (OVA) Protein Particles

Ovalbumin (OVA, 200 mg) was dissolved in distilled water (10 ml) to forma 2% protein solution. Lactic acid (100 μl) was added to theOVA-solution until the pH was reduced to about 4.5-5.0. The solution wasstirred over a magnetic stirrer at 500 rpm for 10 minutes. Acetone (25ml) was added to the stirring solution, and the mixture was leftstirring for 10 minutes. The mixture was heated at 70° C. and stirred at500 rpm for 30 minutes to stabilize the protein particles. The proteinparticles were lyophilized and then sized in a Malvern Master sizer andstored in a dessicator for future use (the protein particles were about2.5 μm in diameter).

Example 3

Preparation of Small gB2 Protein Particles

HSVgB2 antigen (4.2 mg) was dissolved in distilled water (2 ml), and thesolution was stirred over a magnetic stirrer at 1500 rpm. Acetone (2.5ml) was added to the stirring solution, and the mixture was leftstirring for 20 minutes. The mixture was then heated at 70° C. and leftstirring for 25 minutes to stabilize the protein particles. The mixturewas centrifuged at 30,000×g and the protein particles were collected.The particles were lyophilized and then sized in a Malvern Master sizerfor future use (the protein particles were about 350 nm in diameter).

Example 4

Preparation of Large gB2 Protein Particles

HSVgB2 antigen (4.2 mg) was dissolved in distilled water (2 ml), and thesolution was stirred over a magnetic stirrer at 750 rpm. Acetone (2.5ml) was added to the stirring solution, and the mixture was leftstirring for 20 minutes. The mixture was then heated at 70° C. and leftstirring for 25 minutes to stabilize the protein particles. The mixturewas centrifuged at 30,000×g and the protein particles were collected.The protein particles were lyophilized and then sized in a MalvernMaster sizer for future use (the protein particles were about 5 μm indiameter).

Example 5

Preparation of PLG Particles

PLG (poly(lactideco-glycolides)) particles were prepared using polyvinylalcohol (PVA) as follows. Solutions used were:

-   (1) 66% RG 503 PLG (Boehringer Ingelheim) in dichloromethane    (“polymer solution”);-   (2) 8% polyvinyl alcohol (PVA) (ICN) in water (“PVA solution”)    PLG particles were prepared by combining 10 ml of polymer solution    with 40 ml of the PVA solution and homogenizing for 3 minutes using    an Omni benchtop homogenizer with a 10 mm probe at 10 K rpm. The    emulsion was left stirring overnight for solvent evaporation. The    formed PLG particles were washed with water by centrifugation 4    times, and lyophilized. The PLG particles were then sized in a    Malvern Master sizer for future use.

Example 6

Preparation of PLG OVA-Entrapped Particle Using A Solvent EvaporationTechnique

In a 15 ml glass test tube were placed 1 ml of 10 mg/m OVA and 20 ml of5% w:w PLG (poly D,L-lactide-co-glycolide) in dichloromethane, 50:50 molratio lactide to glycolide, MW average=70-100 kDa, (MedisorbTechnologies International). The solution was homogenized for 2 minutesat high rpm using a hand held homogenizer. The homogenate was added to80 ml of 10% polyvinyl alcohol (PVA) (12-23 kDa) in a 100 ml glassbeaker. This was homogenized for two minutes at a 10,000 rpm using abench scale homogenizer equipped with a 20 mm diameter generator. Thesolution was stirred at room temperature at a moderate rate using amagnetic stir bar until the solvents were evaporated. PLG OVA-entrappedparticles were resuspended in water and washed several times with water,using centrifugation to pellet the particles between washes. Theparticles were dried in the presence of desiccant (Dririte CaSO₄) undervacuum. Mean volume size was determined to be 0.9 μm by laserdiffraction measurement. Protein content of the PLG OVA-entrappedparticles was determined to be 0.8% w:w by amino acid compositionalanalysis.

Example 7

Immunogenicity of Ovalbumin (OVA) Particles

Ovalbumin, PLG/OVA-entrapped particles, small OVA-protein particles (250nm) and large OVA-protein particles (2500 nm), produced as describedabove, were administered subcutaneously to mice (dose=10 μg). Theanimals were boosted at 14 and 28 days. Serum was collected two weeksfollowing the last immunization and CTL activity assayed as described inDoe et al., Proc. Natl. Acad. Sci. (1996) 93:8578-8583.

Lymphocyte cultures were prepared as follows. Spleen cells (sc) fromimmunized mice were cultured in 24-well dishes at 5×10⁶ cells per well.Of those cells, 1×10⁶ were sensitized with synthetic epitopic peptidesfrom EG7 (EL4 transfected with ovalbumin) and EL4 proteins at aconcentration of 10 μM for 1 hour at 37° C., washed, and cocultured withthe remaining 4×10⁶ untreated sc in 2 ml of culture medium [50% RPMI1640 and 50% alpha-MEM (GIBCO)] supplemented with heat-inactivated fetalserum, 5×10⁻⁵ M 2-mercaptoethanol, antibiotics, and 5% interleukin-2(Rat T-Stim, Collaborative Biomedical Products, Bedford, Mass.). Cellswere fed with 1 ml of fresh culture medium on days 3 and 5, andcytotoxicity was assayed on day 6.

The cytotoxic cell assay was conducted as follows. EG7 (EL4 transfectedwith ovalbumin) and EL4 target cells used in the ⁵¹Cr release assaysexpress class I but not class II MHC molecules. Approximately 1×10⁶target cells were incubated in 200 μl of medium containing 50 μCi (1Ci=37 Gbq) of ⁵¹Cr and synthetic Ovalbumin peptides (1 μm) for 60 minand washed three times. Effector (E) cells were cultured with 5×10³target (T) cells at various EST ratios in 200 μl of culture medium in96-well round-bottom tissue culture plates for 4 hours. The average cpmfrom duplicate wells was used to calculate percent specific ⁵¹Crrelease.

The small and large OVA-protein particles elicited a CTL response andthe small OVA-protein particles had activity comparable to the largeOVA-protein particles. Both types of OVA-protein particles were moreactive, e.g., elicited a greater immune response than thePLG/OVA-entrapped particles and ovalbumin alone formulations.

Example 8

Preparation of PLG gB2-Entrapped Particle Using A Solvent EvaporationTechnique

In a 15 ml glass test tube was placed 0.5 ml 5 mg/ml gB2 and 5 ml 6% w:wPLG (poly D,L-lactide-co-glycolide) in dichloromethane, 50:50 mol ratiolactide to glycolide, MW average=70-100 kDa, (Medisorb TechnologiesInternational). The solution was homogenized for 2 minutes at high rpmusing a hand held homogenizer. The homogenate was added to 20 ml 8%polyvinyl alcohol (PVA) (12-23 kDa) in a 100 ml glass beaker. Themixture was homogenized for two minutes at a 10,000 rpm using a benchscale homogenizer equipped with a 20 mm diameter generator, The solutionwas stirred at room temperature at a moderate rate using a magnetic stirbar until the solvents were evaporated. PLG gB2-entrapped particles wereresuspended in water and washed several times with water, usingcentrifugation to pellet the particles between washes. The particleswere dried in the presence of desiccant (Dririte CasO₄) under vacuum.Mean volume size was determined to be 0.9 μm by laser diffractionmeasurement. Protein content of the PLG gB2-entrapped particles wasdetermined to be 0.5% w:w by amino acid compositional analysis.

Example 9

Immunogenicity of gB2 Particles

The gB2 protein particles, PLG gB2-entrapped particles, produced asdescribed above, as well as gB2 alone, without associated proteinparticles (as a negative control) and vaccinia gag-pol controls (as apositive control) were administered subcutaneously to mice (dose=5 μg).The animals were boosted at 7 and 14 days. Serum was collected two weeksfollowing the last immunization and CTL activity assayed as described inDoe et al., Proc. Natl. Acad. Sci. (1996) 93:8578-8583.

The lymphocyte cultures were prepared as follows. Spleen cells (sc) fromimmunized mice were cultured in 24-well dishes at 5×10⁶ cells per well.Of those cells, 1×10⁶ were sensitized with synthetic epitopic peptidesfrom HIV-1_(SF2) proteins at a concentration of 10 μM for 1 hour at 37°C., washed, and cocultured with the remaining 4×10⁶ untreated sc in 2 mlof culture medium [50% RPMI 1640 and 50% alpha-MEM (GIBCO)] supplementedwith heat-inactivated fetal calf serum, 5×10⁻⁵ M 2-mercaptoethanol,antibiotics, and 5% interleukin 2 (Rat T-Stim, Collaborative BiomedicalProducts, Bedford, Mass.). Cells were fed with 1 ml of fresh culturemedium on days 3 and 5, and cytotoxicity was assayed on day 6.

The cytotoxic cell assay was conducted as follows. SvBALB (H-2^(d))(SvB) and MCS7 (H-2^(b)) target cells used in the ⁵Cr release assaysexpress class I but not class II MHC molecules. Approximately 1×10⁶target cells were incubated in 200 μl of medium containing 50 μCi (1Ci=37 Gbq) of ⁵¹Cr and synthetic HIV-1 peptides (1 mM) for 60 min andwashed three times. Effector (E) cells were cultured with 5×10³ target(T) cells at various E/T ratios in 200 μl of culture medium in 96-wellround-bottom tissue culture plates for 4 hours. The average cpm fromduplicate wells was used to calculate percent specific ⁵¹Cr release.

The gB2 protein particles were less active than the vaccinia control andwere more active than the PLG/gBf2-entrapped particles and the gB2protein formulation (data not shown).

Example 10

Preparation of Virus Envelope Protein (gp120) With an Adjuvant (R848) onPolymer Microparticles for Administration to Mice

Microparticles were prepared by a solvent evaporation technique byhomogenizing 10 ml of 6% w/v polymer solution in methylene chloride with2.5 ml PBS using a 10-mm probe (Ultra-Turrax T25 IKA-Labortechnik,Germany) for 2 minutes, thus forming a water in oil emulsion. Thisemulsion was then added to 50 ml of distilled water containing 6 ug/mlDSS and homogenized at 15,000 rpm using a 20-mm probe (ES-15 OmniInternational, GA, USA) for 25 minutes in an ice bath, resulting in theformation of water in oil in water emulsion which was stirred at 1000rpm for 12 hours at room temperature, and the methylene chloride wasallowed to evaporate.

A suspension containing 100 mg of PLG was incubated with 1 mg gp120protein and 1 mg R848 in 10 ml total volume PBS. The suspension was thenagitated on a lab rocker (Aliquot Mixer, Miles Laboratories) at 4° C.overnight. The suspension was then aliquoted into small glass vials andlyophilized with manitol at 5.5% wt/v.

Lyophilized product was resuspended in water for injection by brieflyvortexing and administered by intra-muscular injection into mice, with50 μL given into each leg, resulting in a total of 100 μL containing 10μg of the antigen env gp120, 1 mg of RG503(PLG), and the formulatedadjuvants at the dosages described.

FIGS. 1-2 demonstrate that the compositions of the present inventionhave an inhibitory effect on induction of antigen (Ag)-specificIFNγ-secreting cells, while having no inhibitory or stimulatory effecton the induction of antigen-specific IL4-secreting cells. Data is shownfor antigens coadministered with R-848 and/or PLG's and/or MF59. Alsoshown are data with PLGs comprising the MenB ORF287 antigen with PLG andCpG (FIG. 3).

Thus, novel compositions and methods for using and making the same aredisclosed. While the present invention has been described with referenceto the specific embodiments thereof, it should be understood by thoseskilled in the art that various changes may be made and equivalents maybe substituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe subject matter described herein.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

1. An immunogenic composition comprising a first antigen, at least twoadjuvants, wherein a first adjuvant comprises a polymer derived frompoly(lactides) and/or poly(lactide-co-glycolides), and wherein a secondadjuvant comprises an imidazoquinoline, wherein said first antigen isencapsulated within, adsorbed or conjugated to, co-lyophilized or mixedwith said first adjuvant, and a pharmaceutically acceptable excipient,wherein said composition elicits a cellular immune response whenadministered to a vertebrate subject.
 2. The immunogenic composition ofclaim 1 wherein said first antigen is in the form of a particle.
 3. Theimmunogenic composition of claim 1 wherein said first antigen is anucleic acid molecule.
 4. The immunogenic composition of claim 3 whereinsaid nucleic acid molecule is DNA or RNA.
 5. The immunogenic compositionof claim 3 wherein said nucleic acid molecule is linked to a regulatorysequence which controls expression of said nucleic acid molecule.
 6. Theimmunogenic composition of claim 1 wherein said first antigen is aprotein.
 7. The immunogenic composition of claim 1 further comprising atleast a second pharmaceutically acceptable excipient.
 8. The immunogeniccomposition of claim 6 wherein said protein is selected from the groupconsisting of a viral, fungal, bacterial, avian or mammalian protein. 9.The immunogenic composition of claim 8 wherein the viral protein is aherpes simplex virus type 2, hepatitis C virus (HCV), meningococcalantigen, or a human immunodeficiency virus (HIV) protein.
 10. Theimmunogenic composition of claim 9 wherein said HCV protein is E1E2polypeptide.
 11. The immunogenic composition of claim 9 wherein saidmeningococcal antigen is a MenB protein from ORFs 287 and/or 961 ofMenB.
 12. The immunogenic composition of claim 8 wherein the protein isovalbumin, HSVgB2, gp120, p55gag, or combinations thereof.
 13. Theimmunogenic composition of claim 1 wherein at least one adjuvant ispresent in an amount from about 0.01% to about 2% w/v.
 14. Theimmunogenic composition of claim 13 wherein at least one adjuvant ispresent in an amount from about 0.06% to about 1% w/v.
 15. Theimmunogenic composition of claim 14 wherein at least one adjuvant ispresent an amount from about 0.1% to about 0.6% w/v.
 16. The immunogeniccomposition of claim 1 wherein the composition comprises R-848.
 17. Theimmunogenic composition of claim 1 further comprises at least a secondadjuvant comprising MF59.
 18. The immunogenic composition of claim 16comprising a conventional adjuvant.
 19. The immunogenic composition ofclaim 1 further comprising a conventional adjuvant.
 20. The immunogeniccomposition of any one of claims 18 or 19 wherein the conventionaladjuvant comprises aluminum salts, MF59, SAF, Ribi™ adjuvant system(RAS), saponin adjuvants, Complete Freunds Adjuvant (CFA), andIncomplete Freunds Adjuvant (IFA), cytokines, macrophage colonystimulating factor (M-CSF), tumor necrosis factor (TNF), VEGF, CD27,CD30, CD40, Fas Ligand, Placenta Growth Factor, detoxified mutants of abacterial ADP-ribosylating toxin such as a cholera toxin (CT), apertussis toxin (PT), or an E. coli heat-labile toxin (LT), Cpgadjuvants, PT-K9/G129, or combinations thereof.
 21. The immunogeniccomposition of claim 1 further comprising a second antigen distinct fromthe first antigen.
 22. The immunogenic composition of claim 21 whereinthe second antigen is a soluble or neutralizing antigen.
 23. Theimmunogenic composition of claim 21 wherein said second antigen is aprotein or a nucleic acid molecule.
 24. The immunogenic composition ofclaim 21 wherein the second antigen is conjugated to an adjuvant. 25.The immunogenic composition of claim 21 wherein the second antigen isassociated with a carrier.
 26. The immunogenic composition of claim 24wherein the carrier is a protein, polysaccharide, a polylactic acid, apolyglycolic acid, a polymeric amino acid, an amino acid copolymer, alipid aggregate, a polymeric particulate carrier, or an inactive virusparticle.
 27. The immunogenic composition of claim 26 wherein thepolymeric particulate carrier comprises a polymer selected from thegroup consisting of a poly(α-hydroxy acid), a polyhydroxy butyric acid,a polycaprolactone, a polyorthoester, and a polyanhydride.
 28. Animmunogenic composition comprising a first antigen, at least twoadjuvants, wherein a first adjuvant comprises a polymer derived frompoly(lactides) and/or poly(lactide-co-glycolides), and wherein a secondadjuvant comprises an imidazoquinoline, and a pharmaceuticallyacceptable excipient, wherein the first antigen is a particle producedby a process comprising the steps of: (a) adding a precipitation agentto an aqueous solution of an antigen and stirring the resulting mixtureto form the particle; (b) stabilizing the antigen particle by astabilizing treatment; and (c) recovering the antigen particle from theaqueous solution.
 29. The immunogenic composition of claim 28 whereinsaid first antigen is a protein or a nucleic acid molecule.
 30. Theimmunogenic composition of claim 28 wherein said imidazoquinoline isR-848.
 31. The immunogenic composition of claim 28 wherein theimmunogenic composition further comprises MF59.
 32. The immunogeniccomposition of claim 28 further comprising a second pharmaceuticallyacceptable excipient.
 33. The immunogenic composition of claim 28wherein the aqueous solution in step (a) further comprises an acid. 34.The immunogenic composition of claim 28 wherein the process comprises asolvent evaporation technique.
 35. The immunogenic composition of claim33 wherein the acid is one or more of acetic acid, glycolic acid,hydroxybutyric acid, hydrochloric acid or lactic acid.
 36. Theimmunogenic composition of claim 28 wherein the precipitation agentcomprises one or more of oils, hydrocarbons or coacervation agents. 37.The immunogenic composition of claim 28 wherein the stabilizingtreatment comprises one or more of heat treatment or treatment with achemical cross-linking agent.
 38. The immunogenic composition of claim28 wherein the particle elicits a cellular immune response in avertebrate subject and is formed from a protein selected from the groupconsisting of a tumor, a viral, a fungal, a bacterial, an avian or amammalian protein.
 39. The immunogenic composition of claim 28 whereinthe vertebrate subject is a human.
 40. The immunogenic composition ofclaim 38 wherein the protein is a herpes simplex virus type 2 protein,hepatitis C virus (HCV) protein, an influenza A virus protein, or ahuman immunodeficiency virus (HIV) protein, or an immunogenic fragmentof a herpes simplex virus type 2 protein, a hepatitis C virus (HCV)protein, an influenza A virus protein, or a human immunodeficiency virus(HIV) protein, or combinations thereof.
 41. The immunogenic compositionof claim 40 wherein the protein is E1E2 polypeptide, HSVgB2, gp120,p55gag, MenB protein from ORFs 287 and/or 961, immunogenic fragments ofE1E2 polypeptide, HSVgB2 or gp120, p55gag, MenB protein from ORFs 287and/or 961, or combinations thereof.
 42. The immunogenic composition ofclaim 38 wherein the bacterial protein is pertussis protein, adiphtheria protein, a meningitis protein, a H. pylori protein, a H.influenza B protein, or a tetanus protein, immunogenic fragments of apertussis protein, a diphtheria protein, a meningitis protein, a H.pylori protein, a H. influenza B protein, or a tetanus protein, orcombinations thereof.
 43. The immunogenic composition of claim 38wherein the cellular immune response is a cytotoxic-T lymphocyte (CTL)response.
 44. The immunogenic composition of claim 2 or claim 28 whereinthe immunogenic composition further comprises a second antigen andwherein the particle functions as an antigen and/or an adjuvant.
 45. Theimmunogenic composition of any one of claims 9 or 40 wherein the HCVantigen is HCV core protein, E1, E2, NS3, NS4, or NS5, or an immunogenicfragment of HCV core protein, E1, E2, NS3, NS4, or NS5, or combinationsthereof.
 46. The immunogenic composition of any one of claims 9 or 40wherein the HIV protein is gp120, gp160, gp41, p24gag or p55gag, or afragment of gp120, gp160, gp41, p24gag or p55gag, or combinationsthereof.
 47. The immunogenic composition of claim 28 wherein thestabilized particle is generally spherical.
 48. The immunogeniccomposition of claim 28 wherein the stabilized particle has a diameterfrom about 200 nanometers to about 10 microns.
 49. The immunogeniccomposition of claim 28 wherein the stabilized particle has a diameterranging from about 500 nanometers to about 5 microns.
 50. A method foreliciting a cytotoxic-T lymphocyte (CTL) response in a vertebratesubject comprising administering to the vertebrate subject animmunogenic composition of any one of claims 1 or
 28. 51. The method ofclaim 50 wherein the vertebrate subject is a human.
 52. The method ofclaim 50 wherein the immunogenic composition is co-administered to thesubject prior or subsequent to, or concurrent with, an adjuvant and/or asecond antigen.
 53. A method of eliciting an immune response in avertebrate subject comprising administering to the vertebrate subject aeffective amount of the immunogenic composition according to any one ofclaims 1 or
 28. 54. The method of claim 52 wherein the vertebratesubject is a human.
 55. The method of claim 50 wherein the immunogeniccomposition is administered by a route of intramuscular, intratracheal,intranasal, transdermal, intradermal, subcutaneous, intraocular,vaginal, rectal, intraperitoneal, intraintestinal or inhalationadministration.
 56. The method of claim 52 wherein the immunogeniccomposition is administered by a route of intramuscular, intratracheal,intranasal, transdermal, intradermal, subcutaneous, intraocular,vaginal, rectal, intraperitoneal, intraintestinal or inhalationadministration.
 57. The method of claim 50 wherein the immunogeniccomposition is delivered by a device selected from the group consistingof a particle accelerator, a pump, an intradermal applicator, abiolistic injector, a pneumatic injector, a sponge depot, a pill, and atablet.
 58. The method of claim 52 wherein the immunogenic compositionis delivered by a device selected from the group consisting of aparticle accelerator, a pump, an intradermal applicator, a biolisticinjector, a pneumatic injector, a sponge depot, a pill, and a tablet.59. The method of claim 50 wherein the immunogenic composition isdelivered by needle-free injection.
 60. The method of claim 52 whereinthe immunogenic composition is delivered by needle-free injection. 61.An injectable vaccine composition comprising an immunogenic compositionof any one of claims 1 or
 28. 62. The injectable vaccine of claim 61wherein the vaccine is administered by via intramuscular injection. 63.The injectable vaccine of claim 61 wherein the vaccine is administeredvia a needle-free injection device.
 64. A method of eliciting anantibody-mediated immune response in an individual comprisingadministering the immunogenic composition of any one of claims 1 or 28to said individual.
 65. A method of making the immunogenic compositionof any one of claims 1 or 28 comprising: (a) combining a precipitationagent with an aqueous solution comprising an antigen; (b) dispersing theresultant mixture to form antigen particles; (c) stabilizing thedispersed antigen particles by a stabilizing treatment; (d) recoveringthe stabilized antigen particles; and (e) combining the stabilizedantigen particles with a pharmaceutically acceptable excipient.
 66. Themethod of claim 65 wherein said antigen is a protein or a nucleic acidmolecule.
 67. The method of claim 65 wherein the process furthercomprises a solvent evaporation technique.
 68. A stabilized particlecapable of eliciting a cytotoxic-T lymphocyte (CTL) response, whereinthe stabilized particle is a generally spherical particle that isproduced by a process comprising: (a) forming a particle from antigen;(b) stabilizing the particle by a stabilizing treatment, wherein thestabilized particle is not a virus-like particle, and wherein thestabilized particle is not entrapped within a carrier; and (c) adding anadjuvant comprising R-848.
 69. The stabilized particle of claim 68wherein said antigen is a protein or a nucleic acid molecule.
 70. Thestabilized particle of claim 68 wherein the process comprises a solventevaporation technique.
 71. The particle of claim 68 wherein thestabilizing treatment is one or more of a heat treatment process or achemical cross-linking process.
 72. The particle of claim 68 wherein theparticle is formed by a precipitation process.
 73. The particle of claim72 wherein the precipitation process comprises combining an aqueousprotein solution with one or more of an oil, a hydrocarbon or acoacervation agent.
 74. The particle of claim 72 wherein the particle isformed by an emulsion process.
 75. The particle of claim 68 wherein thestabilized particle is formed from a tumor protein antigen, a viralprotein antigen, or a bacterial protein antigen.
 76. The particle ofclaim 75 wherein the viral protein is a herpes simplex virus type 2protein, hepatitis C virus (HCV) protein, an influenza A virus protein,or a human immunodeficiency virus (HIV) protein, or an immunogenicfragment of a herpes simplex virus type 2 protein, a hepatitis C virus(HCV) protein, an influenza A virus protein, or a human immunodeficiencyvirus (HIV) protein, or combinations thereof.
 77. The particle of claim75 wherein the bacterial protein is a pertussis protein, a diphtheriaprotein, a meningitis protein, a H. pylori protein, a H. influenza Bprotein, or a tetanus protein, or immunogenic fragments of a pertussisprotein, a diphtheria protein, a meningitis protein, a H. pyloriprotein, a H. influenza B protein, or a tetanus protein, or combinationsthereof.
 78. The particle of claim 76 wherein the protein is HSVgB2,gp120, HCV core protein, E1, E2, NS3, NS4, or NS5, or an immunogenicfragment of HCV core protein, E1, E2, NS3, NS4, NS5, HSVgB2 or gp120, orcombinations thereof.
 79. A method of preparing an immunogeniccomposition comprising combining the stabilized particle of claim 68with one or more pharmaceutically acceptable excipients.
 80. The methodof claim 79 further comprising providing an additional antigen withinthe immunogenic composition, wherein the additional antigen is distinctfrom the antigen particle.
 81. The method of claim 79 wherein thestabilized particle has a diameter from about 200 nanometers to about 10microns.
 82. The method of claim 79 wherein the stabilized particle hasa diameter from about 500 nanometers to about 5 microns.
 83. Apharmaceutical composition comprising an immunogenic composition of anyone of claims 1 or
 28. 84. A pharmaceutical composition comprising astabilized particle of claim
 68. 85. The pharmaceutical composition ofclaim 84 wherein the stabilized particle further comprises aconventional adjuvant.
 86. A kit for preparing an immunogeniccomposition of claim 1 comprising a first container comprising anantigen, a second container comprising an imidazoquinoline, and a thirdcontainer comprising a polymer derived from poly(lactides) and/orpoly(lactide-co-glycolides).
 87. The kit of claim 86 wherein saidimidazoquinoline comprises R-848.
 88. The kit of claim 86 wherein theantigen is a tumor protein, a viral protein, a bacterial protein, afungal protein, an avian protein, or a mammalian protein, or fragmentsof a viral protein, a bacterial protein, a fungal protein, an avianprotein, or a mammalian protein.